CA3138758A1 - Highly soluble leptins - Google Patents

Highly soluble leptins Download PDF

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Publication number
CA3138758A1
CA3138758A1 CA3138758A CA3138758A CA3138758A1 CA 3138758 A1 CA3138758 A1 CA 3138758A1 CA 3138758 A CA3138758 A CA 3138758A CA 3138758 A CA3138758 A CA 3138758A CA 3138758 A1 CA3138758 A1 CA 3138758A1
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seq
leptin
amino acids
amino acid
sequence
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Mary Erickson
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Amylin Pharmaceuticals LLC
AstraZeneca Pharmaceuticals LP
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Amylin Pharmaceuticals LLC
AstraZeneca Pharmaceuticals LP
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2264—Obesity-gene products, e.g. leptin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Abstract

The disclosure provides chimeric polypeptides and nucleic acid molecules encoding chimeric polypeptides. Also provided are pharmaceutical compositions and methods of treatment for diseases and disorders including lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease or diabetes (including type I and type II). Additional diseases and disorders which can be treated by the compounds and methods described herein include non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease (NAFLD), metabolic syndrome X and Huntington's Disease.

Description

=
HIGIIIN SOLUBLE LEPTINS
=
boot' SEQUENCE LISTING
10001.11 The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web. Said ASCII
copy, created on October 27, 2011, is named 1317W01.txt and is 159,244 bytes in size.
BACKGROUND OF THE INVENTION
100021 The invention provides novel compounds that have demonstrated biological activity.
The compounds also demonstrate surprising and significant improvement in physical properties, such as solubility and stability.
100031 The compounds of the invention are based on leptin sequences.
The compounds are surprisingly highly soluble and do not demonstrate the propensity to aggregate, unlike the naturally occurring leptins. The physical properties of the compounds facilitate the preparation of soluble, pharmaceutically acceptable formulations and compositions, also provided by the invention. Diseases amenable to such treatment include lipodystrophy, dyslipidernia, hyperlipideinia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease: diabetes (including type I and type II), nonalcoholic steatohepalitis (NASH), nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X, and Huntington's Disease, or combinations thereof 100041 There remains a need to develop polypeptides useful in the above described metabolic diseases, conditions and disorders. Accordingly, it is an object of the present invention to provide novel polypeptides useful to treat the above conditions and methods for producing and using them.
1 =
Date Recue/Date Received 2021-11-12 -BRIEF SUMMARY OF THE INVENTION
100061 There are. provided chimeric polypeptide compounds having leptin biological activity, in addition to enhanced physical properties. The compounds are chimeric polypeptides which are based on a wild type seal leptin polypeptide wherein at least one contiguous region of 1-30 amino acids of a wild type seal leptin sequence has been replaced with a contiguous region of I-n amino acids of a mature human leptin sequence.
[0007] In a first aspect, there is provided a chimeric polypeptide as described herein.
[00081 In another aspect, there is provided a method for treating a disease or disorder in a subject in need of treatment. The method includes administering a chimeric polypeptide as described -herein to the subject.
100091- In yet another aspect, there is provided a pharmaceutical composition which includes a chimeric polypeptide described herein in combination with a pharmaceutically acceptable excipient.
100.101 In yet another aspect are polynucleotides encoding the chimeric .polypeptide and their intermediates, expression vectors bearing such polynuelcondes, host cells expressing such polynucleotides, and means for their expression, synthesis, post-translational modification and isolation.
BRIEF DESCRIPTION OF THE DRAWINGS
100111 Figs. 1A-1C depict the effects of a daily administration of the indicated chimeric polypeptides described herein on food intake and change in body weight (%
vehicle-corrected) upon administration to C57/86 female mice as described in Example 4. Figure IA: food intake.
Figure I B: change in body weight. (% vehicle-corrected). Figure IC: dose response curve.
[00121 Figs. 2A-2C depict the effects of a daily administration of the indicated chimeric polypeptides described herein on food intake and change in body. weight (%
vehicle-corrected)
2 Date Recue/Date Received 2021-11-12 = upon administration to C57/136 female mice. as described in Example 5.
Figure 2A: food intake.
Figure 2B: change in body weight (% vehicle-corrected): Figure 2C: dose response curve.
DETAILED DESCRIPTION OF THE INVENTION
Definitions 100131 "Obesity" and "overweight" refer to mammals having a weight greater than normally expected, and may be determined by, e.g., physical appearance, body mass index (BMI) as known in the an, waist-to-hip circumference ratios, skinfold thickness, waist circumference, and the like. The Centers for Disease Control and Prevention (CDC) define overweight as an adult human having a BMI of 25 to 29.9; and define obese as an adult human having a BMI of 30 or higher. Additional metrics for the determination of obesity exist. For example, the CDC states that a person with a waist-to-hip ratio greater than 1.0 is overweight.
100141 "Lean body mass" refers to the fat-free mass of the body, i.e., total body weight minus body fat weight is lean body mass. Lean body mass can be measured by methods such as -hydrostatic weighing, computerized chambers, dual-energy X-ray absorptiometry, skin calipers, magnetic resonance imaging (MRI) and bioelectric impedance analysis (BIA) as known in the art. =
=
100151 "Mammal" refers to warm-blooded animals that generally have fur or hair, that give live birth to their progeny, and that feed their progeny with milk. Mammals include humans;
companion animals (e.g., dogs, cats); farm animals (e.g., cows, horses, sheep, pigs, goats); wild animals; and the like. In one embodiment, the mammal is a female. In one embodiment, the mammal is a female human. In one embodiment, the inanunal is a cat or dog. In one = embodiment, the mammal is a diabetic mammal, e.g., a human having type 2 diabetes. In one embodiment, the mammal is an obese diabetic mammal, e.g., an obese mammal having type 2 diabetes. The term "subject" in the context of methods described herein refers to a mammal. =
100161 "Fragment" in the context of Polypeptides refers herein in the customary chemical sense to a portion of a polypeptide. For example, a fragment can result from N-terminal deletion or C-terminal deletion of one or more residues of a parent polypeptide, andlor a fragment can result from internal deletion of one or nmre residues of a parent polypeptide.
"Fragment" in. the
3 =
Date Recue/Date Received 2021-11-12 context of an antibody refers to a portion of an antibody which can be linked to a biologically active molecule to modulate solubility, distribution within a subject, and the like. For example, leptin A200 described herein is a conjugate of an Fe antibody fragment with a leptin, as known in the art. See e.g. WO 98/28427 and US2007/002084. The term "parent" in the context of polypeptides refers, in the customary sense, to a polypeptide which serves as a reference structure prior to modification, e.g., insertion, deletion and/or substitution.
100171 "Analog" as used herein in the context of polypeptides refers to a compound that has insertions., deletions and/or substitutions of amino acids relative to a parent compound. Ait analog may have superior stability, solubility, efficacy, half-life, and the like. Irk some embodiments, an analog is a compound having at least 50%, for example. 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or even higher, sequence identity to the parent compound.
100181 "Identity," "sequence identity" and the like in the context of comparing two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or-nucleotides that are the same (i.e., about 50% identity, preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region. when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a sequence comparison algorithms as known in the art, for example BLAST or BLAST 2Ø This definition includes sequences that have deletions andfor additions, as well as those that have substitutions, as well as naturally occurring, e.g., polymorphic or allelic variants, and man-made variants. In preferred algorithms, account is made for gaps and = the like, as known in the art. For sequence comparison, typically one.
sequence acts as a reference sequence, to which test sequences arc compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequent coordinates are = designated if necessary, and :sequence algorithm program parameters are designated. Preferably, default program parameters can he used, or alternative parameters can he designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm
4 Date Recue/Date Received 2021-11-12 . .
of Smith & Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method or Pearson & Lipman, 1988,Proc. Nan. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science.Dr., Madison, Wis.:), or by manual alignment and visual inspection. See e.g., Current Protocols in Molecular Biology (Ausubel et at., eds. 1995 supplement)). Preferred examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST
2.0 algorithms, which are described in Altschul et al., 1977, Nucl, Acids Res.
25:3389-3402 and Altschul et at., 1990,1. Mol, Biol. 215:403-410. BLAST and-BLAST 2.0 are used, as known in the art, to determine percent sequence identity for the nucleic acids and proteins of the invention.
Software for pertbnning BLAST analyses is publicly available through the web site oldie National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (BSI's) by identifying short words of length Win the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood ward score threshold (Altschul et at., 1d). These initial neighborhood word hits act as seeds for initiating searches to find longer liSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, e.g., for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; alwayt>0) and N (penalty score for mismatehingresidues; always<0).
For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension =
of the word hits in each direction are halted when: the cumulative alignment score falls off by thc quantity X front its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation. of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of II, an expectation (E) of 10, M=5, N--=-4 and a comparison of both strands.
For amino acid sequences, the BLAST? program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Flenikoff & Her-Aar, 1989, Date Recue/Date Received 2021-11-12 Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, and a comparison of both strands.
[00191 The term "about" in the context of a numeric Value refers to +/- 10% of the numeric value, unless expressly indicated otherwise.
[00201 The terms "peptide" and "polypeptide" in the context of the compounds described herein are synonymous.
[0021 J Leptins. "Leptins" and "a leptin" means: leptins, leptin active fragments, leptin analogs, and leptin derivatives; and a leptin, a leptin active fragment, a leptin analog, and a leptin derivative; respectively. Accordingly, unless otherwise noted, reference to "leptins" is meant to encompass leptins, leptin active fragments, leptin analogs, and leptin derivatives, as disclosed herein. Similarly, unless otherwise noted, reference to "a leptin" is meant to encompass a leptin, a leptin active fragment, a leptin analog, and a leptin derivative, as disclosed herein. Exemplary leptins which may be employed in the design, preparation, and use of the chimeric pelypeptides disclosed herein include those which elicit one or more biological responses known in the art to be elicited when leptins are administered to subjects (see, e.g., published U.S. Patent application Nos. US 2007/0020284 and US 2008/0207512, U.S. Patent Nos. 6,309,853, and US
7,183,254, and PCT Published Application Nos. WO 96/005309, WO 98/28427, and WO
2009/064298), such as: reduction of food intake, reduction of body weight, reduction of body weight gain, induction of satiety, reduction of calorie availability, reduction of caloric efficiency, reduction of metabolic plateau, increase in insulin sensitivity, reduction of hyperlipidemia, correction of dyslipidetnia, reduction of hypertriglyeeridemia, amelioration of obesity, amelioration of overweight, amelioration of diabetes mellitus (including type I diabetes, type II diabetes, and gestational diabetes), amelioration of insulin resistance, amelioration of lipodystrophy conditions . associated therewith, as well as other biological responses known in the art to be elicited upon administration of a leptin (see, e.g., published U.S. Patent Application Nos.

and US 2008/0207512, U.S. Patent Nos. 6,309,853, and US 7,183,254, and PCT
Published Application Nos. WO 961005309, WO 98/28427, and WO 2009/064298).
100221 Leptins include, but are not limited to, the compounds described in U.S. Patent Nos.
US 5,594,101, US 5,851,995, US 5,691,309, US 5,580,954, US 5,554,727, US
5,552,523, US 5,559,208, US 5,756,461, US 6,309,853, published U.S. Patent application No. US
6 Date Recue/Date Received 2021-11-12 2007/0020284, and PCT Published Application Nos, WO 96/23517, WO 96/0115309, WO
98/28427, WO 2004/039832, WO 98/55139, WO 98/12224, and WO 97/02004.
Methods to assay for leptin activities and biological responses in vitro and in vivo, including satiety, food intake inhibition activity and weight loss activity, are known in the art and are described herein and also in the above references and other references recited herein.
100231 Representative leptins, [coin analogs, leptin active fragments, and leptin derivatives include the following:
100241 Mature Murineteptins:
VPIQKVQDDTKTLIKTIVTRINDISHTAaa-SVSSKQKVTGLDFIPGLHPILTLSKMDQTLA

STEVVALSRLQGSLQDMLQQLDLSPGC, wherein Xaa at position 23 is Q or absent (SEQ ID

NO:1), 100251 Mature Murine leptin form 1:

QQVLTSLPSQNVLQIAN.DLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDGVLEASLYST
EVVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO:2).
100261 Mature Murine leptin form 2:
VPIQKVQDDTKTLIKTIVTRINDISHTSVSAKQRVTGLDFIPGLHPILSLSKMDQTLAVYQ
QVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDG VLEASL YSTE
VVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO:3).
100271 Mature Murine leptins with N-terminal methionine: =
MVPIQKVQDDTKTLIKTIVTRINDISHT-Xaa-SVSSKQKVTGLDFIPGLHPILTLSKMDQTL
AVVQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASCiLETLESLGGVLE.ASG
YSTEVVALSRLQGSLQDMLQQLDLSPGC, wherein Xaa at position 29 is Q or absent (SEQ
ID NO:4). =
100281 Mature Murine leptin form 1 with N-terminal methionine: .
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSAKQRVIGLDFIPGLHPILSLSKMDQTLAV
7 =
Date Recue/Date Received 2021-11-12 =
YQQVUTSLPSQNVLQI.ANDLENIADLLEILLAFSKScSLPQTSGLQKPESLDGVLEASLYS
TEVVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO:).
100291 Mature Murine leptin form 2 with N-terminal methionine:
MVPIQKVQDDT.KTLIKTIVTRINDISHTSVSAKQRVTGLDFIPGIAPILSLSKIvIDQTLAVY
QQVI,ISLPSQNVLQIAN.DLENIADE.I.141.L.AFSKSCSLPQTSG1QKPESI,DGV1EASLYST
EVVAL.SRL.QGS.L.QDILQQ1..DVSPEC (SEQ ID NO:6).
100301 Mature.Porcine leptin:
VPIWRVQDDTKTLIKTIVIRISDISHMQSVSSK.QRVTGI.DFIPG1.11PVLSI:SKMDQTLAIY
= QQ1LISLPSRNVIQISNDLENLRDILHLLASSKSCPLPQARALETLESLGGVLEASLYSTEV
VALSRLQGALQDMLRQLDI_SPGC (SEQ ID NO:7).
[00311 Mature Porcine leptin with N-terminal methionine:

YQQILTSLPSRNVIQISNDLENLIZDLI.ELLASSKSCPLPQARALETLESLGGVLEASLYSTE
VVALSRL.QGALQDMI-.RQLDI.,SPGC (SEQ lID NO:8).
[00321 Mature Bovine Ieptins:
VPICKVQDDIKTLIKTIVTRIND]SHT-Xaa-SVSSKQRVTGLDFIPGLHPLLSLSKMDQUAI
YQQ1LTSLPSRNVVQISNDLENLIUDLLHLLAASKSCHTQVRALESLESLGVVI.EASpesT
EVVALSRLQGSLQDMI-RQLDLSPGC;whercin Xaa at position 28 is Q or absent (SEQ ID
NO:9).
[00331 Mature Bovine leptins with N-terminal methionine:
MVPICKVQDDIKTLIKTIVIRENIDISHT-Xaa-SVSSKQRVTGLDFIPGLHPLLSLSKIADQII
ALYQQ1LTSLIDSRNVVQ1SNDLENLADLLI-ILLAASK.SCP1,13QVRALESLESI,GVVLEASLY
STEVVALSRLQGSLQDIALRQLDLSPGC, wherein Xaa at position 29 is Q or absent (SEQ
ID
N0:10.
= 100341 Unprocessed Full-length Human Leptin (i.e., includes 21-residue N-terminal signal sequence): =
N1HWGILLGETWIMPYLPYVQ.AVPIQKyQDDTKTI,IKTIVITINDISHTQSVSSKQKVTG
LDIIPGLIVILILSKIADQTLAV YQOILTSMPSRNVIQISNDLENLRDLLFIV LAFSKSCH LI)
8 Date Recue/Date Received 2021-11-12 WASGLETLDSLGOVLEASGY STEVVALSRLQ0SLQDIALWQLDLSPGC. (SEQ ID NO:
11) (00351 Mature Human leptins (with N-terminal 21 amino acid signal sequence removed):
VPIQKVQDDTKTLIKTIVIRINDISH-Xaa-Xaa-SVSSKQKVTOLDFIPGLHPILTLSKIADQT
LA.VYQQIILTSMPSRNV1Q1SNDLENLRDLTHVLAFSKSCIILP WASOLETLDSLOGVI...EAS
GYSTEVVALSRLQGSLQDMLWQ.LDLSPOC, wherein: .Xaa at position 27 is 7 or A ; and Xaa at position 28 is Q or absent (SEQ ID NO:12).
(00361 Mature Human lepthis with N-terminal methionine:
MVPIQKVQDDTKIL1KTIVTRINDISH-Xaa-Xaa-SVSSKQKVTGLDFIPOLHPILTLSKIvIDQ

SGYSTEVVALSRLQGSLQDMILWQLDESPOC, wherein: Xaa at position 28.is T or A ; and Xaa at position 29,is Q or absent (SEQ ID NO:13).
10037) Mature Rhesus Leptin:
-VPIQKVQSDTKTLIKTIVTRINDISHTQSVSSKQRVIGLDFIPGLHINLTLSQMDQTLAll'Q
QILINLPSRNVIQISNDLENLRDLUILLAFSKSCHLPLASGLETLESLODVLEASLYSTEVV
ALSRLQGS11.QDMLWQLDLSF.}Ge. (SEQ NO:14).
100381 Mature Rhesus Leptin with N-terminal methionine:
MVPIQKVQSDTKTLIKTIVTRINDISHTQSVSSKQRVTCiLDFIPGLHPVLTLSQMDQTLAI
YQQ11JNLPSRNVIQISNDLENLRDLL1.1.1.1.AFSKSCHLPLASGLETLESLODVLEASLYSTE
VVALSRLQGSLQDIALWQEDLSKIC (SEQ ID NO: 15).
10039.1 Mature Rat leptin:
VPIHKVQDDIX7.1,11(71VTRINDISHTQSVSARQRVTGLDFIPOLIIPILS1SKNIDQUAVY
QQ1LTSLPSQNVLQ1AHDLENLRDLLITLLAFSKSCSL.PQTROLQKPESLDGVLE.ASLYSTE
VVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO:16).
1004(11 Mature Rat leptin with N-terminal methionine:

YQQ1LTSLPSQNVLQI.AH.DLENLRDLLIILL.AFSKSCSLIPQ7R.GLQKPESLDGVLE.ASLYST
EVVALSRLQGSLQUILQQLDLSPE( (SEQ ID NO: I 7).
. 9 Date Recue/Date Received 2021-11-12 100411 Mature Platypus leptin: The mature platypus leptin sequence follows:
ISIEKIQADTKTLTKTIITRUQLSTQNGVSTDQRVSGLDFIPONQQFQNLA.DMDQTLAVYQ
QILSSLPNIPDIZTQISNDLENLRSISALLAILKNCPETRSDOLDTME,IWGGIVEESLYSTEV
VTLDRLRKSLKN1EKQLDHIQG (SEQ ID NO: IS).
100421 Unprocessed Full-length Platypus leptin (i.e., includes 21-residue N-terminal signal sequence): A Full length sequence of platypus leptin, including a 21-residue N-terminal signal sequence follows:
MR.C.ILLYGELCVWQHLYYSEIP1S1EKIQADTKTLTKTI1TRHQLSTQNGVSIDQ11VSGLDF

DOLDTMEIWGGIVEESLYSTEVVTLDRLRKSLKNIEKQLDHIQG (SEQ ID NO:19).
100431 Mature Human Leptin form 1: =
VPIQINQDDIKTLIKTIVTRLNDISHTQSVSSKQKVTGLDFIPGLIIPILTLSKIMDQTLAVY
QQ11,TSMPSRNVIQISNDLENLRDLLIDILAFSKSCHLPWASGLETLDSLOGNILEA SG YST
EVVALSRLQC.iSLQDNII_WQLDLSPGC (SEQ ID NO:20).
100441 Mature Human Leptin form 2:
VPIQKVQDDTKTLIKTIVTRINDISHAQSVSSKQKVTC.iLDFIPGLEIPILTISKMDQTLA VY

EILVALSRLQGSLQDNILWQLDLSPOC (SEQ ID NO:2 I).
100451 Mature Human Leptin form 3:
=VPIQKVQDDTKTLIKTIVTRINDISHTSVSSKQKVTGLDFIPOLHPILTLSICMDQTLAVYQ
QILTSMPSRNVIQ.ISNDLENLRDLLEIVLAFSKSCHLPWASGLETLDSLOGVLEASGYSTE
VVALSRLQGSLQDMLWQL-DLSPOC(SEQ ID NO:22).
=
100461 Mature 'Human Leptin form 4:
VIIQKVQDDTKTLIKTIVTRINDIS.HASVSSKQKVTCiLDFIPOLHPILTLSKIADQTLAVYQ
Q1LTSMPSRNVIQ1S.NDLENLRDLLHVLAFSKSCHILPWASGLETLDSLOGVLEASGYSTE
VVALSRLQGSLODMLWQLDLSPGC (SEQ ID NO:23).
= = 100471 Mature 11 man Leptin form 1 with N-terminal methionine (also known as Metreleptin, or A100):
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAV
Date Recue/Date Received 2021-11-12 =
YQQ1LTS1v1PSRNVIQISNDLENLRDLLHAILAFSKSCHLPWASOLEILDSLOGVLEASGYS
TEVyALSRLQGSLQDNILWQLDLSPGC. (SEQ ID NO:24).
100481 Mature Human Leptin form 2 with N-terminal rnethionine:
MVPIQKVQDDTKTLIKTIVTILINDISHAQSVSSKQKVTGLDFIPGLH:PILTLSKMDQTLAV
YQQ1LTSMPSRNVIQISNDLENLRDI.111:11LAFSKSCHLPWASOLETLDS I,GOVLEA SG YS
TEVVALSRLQGSLQDMLWQLDLSPOC (SEQ ID NO:25). =
100491 Mature Human Leptin form 3 with N-terminal methionine:
MVPIQK.VQDDTKTLIKTIVTRANDISEITSVSSKQKVTGI.DEIPOLI4PILTLS.KMDQTLAVY
= QQ1LISM1SRNVIQISNDLENLRDLLIIVLAFSKSCI ILPWASGLETLDSLGOVILEASGYST
EVVALSRLQGSLQDMLWQLDLSPGC (SEQ. ID NO:26).
(00501 Mature Human Leptin form 4 with N-terminal methionine:
MVPIQKVQDDTKTLIKTIVIRINDISHASVSS KQK VTOL NI PG E.11111...TISK M DOT LA Y
OQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYST
EVVALSRLQGSLQDMLWQLDLSPGC (SEQ ID NO:27). .
100511 Seal leptin:
PIQRVQDDIKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQ
ILTSLQSKSVVQ1ANDLANLRALLRLLASAKSCPVPRARGSDTIKGLONVI.RASVHSTEV
VALSRI,KAALQDMLROLDRNPGC (SEQ ID NO:28). =
100521 Seal leptin with amino acids 71-92 replaced with amino acids 73-94 (helix 3) of metreleptin, respectively:
PIQ RVQDDTKTLIKTIITRIN DISPPQQVCSRPRVAGLDFTPR VQSAIRTLS GNI DQILATYQQ
ILTSLQSRNVIQISNDLENLRDLL EIVLAFSK.SCPVPR.ARGSDTIK.GLONVLRASVIIST.EVV
ALSRLKAALQ.DMLRQLDRNPOC (SEQ. ID 'NO:29).
100531 Seal leptin with amino acids 30 and 71-92 replaced with amino acids 32 and 73-94 (helix 3) of inetreleptin, respectively:
PIQRVQ.DDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSV RT LSO felDQILA TY QQ1 LTSLQSRNVIQISNDLENIADLLHVLAFSKSCPVPRAR.(JSDT1 KGI,ONVI_RAS1/ /ISTEVV
ALSRLKAALQDMLIZQLDRNPC;C (SEQ. I)NO:30).
=

Date Recue/Date Received 2021-11-12 10054! Seal leptin with N-terminal methionine:
MPIQRVQDDIKTLI KTIITRINDISPPQGVCSRPRVAGLDFIPRvQSVRTI,SGNIDQILATYQ
QIUFSLQSR.SVVQ1ANDLANDIALI,RI,LASAKSCPWRARGSDTIKG1...GNVI.RASVIISTE
.VVALSRLKAALQDMLIZQLDRNPGC (SEQ ID NO:31).
(00551 Seal leptin with N-terminal methionine, and with amino acids 71-92 replaced with amino acids 73-94 (helix 3) of metreleptin, respectively:
MPIQRVQDDTKTLIKTITTRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQ
QILTSLQSRNVIQISNDLENE.RDWWLAFSKSC.PVPRARGSDTIK.GLGNVLRASVHSTEV
Ar'ALSRLICAALQDMLRQLDRNPOC. (SEQ ID NO:32).
10056] Seal leptin with N-terminal methionine, and with amino acids 30 and 71-replaced with amino acids 32 and 73-94 (helix 3) of metreleptin, respectively:

MPIQRVQDDTICTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSOMDQILATYQ
QIII:TSLQSRNVIQISNDLENIADLLHVLAPSKSCPVPRARGSIDTIK.GLONVLRASVHSTEV
VALSRLKAALQDMLRQLDRNPOC. (SEQ ID NO:33).
100571 Leptin A200: Leptin A200 is an Fc antibody fragment condensation product with leptin. as known in the art. See e.g., Lo et al., 2005, Protein Eng. Design (..Q Selection, 18:1-10.
The amino acid sequence of A200 is as follows:
MDKTEITCPPCPAPELLGGPSVFLFPPKPKDTLMISRIPEVTC.VVVDVS.1-1EDPEVK.FNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SKAKGQ.PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKETVDKSRWQQGNVFSCSVMHEALliNHYTQKSLSLSPOKVPIQKV "

MPSRNVIQISNDLENLIIDLLIIVLAFSKSCHLPWASGLETLDSLGGVLEASOYSTEVVALS
= RLQGSLQD1vILWQ.LDLSPOC (SEQ ID NO:34) 100581 Leptin A300: Leptin A300 is mehreleptin with substitutions \41101Q and W139Q.
(N-terminal IMet counted as residue I):
MVPIQKVQ.DDTKILIKTIVTRINDISHIQSVSSKQKVTGLDFIPGLHPILTLSKIvIDQTLAV
If LTSM PS RNVI Q ISN DUN I.,RDL LFIV.I.AFS KSCHLPQASC; L ETLDSLOGVLEASGYS

TEVVALSRLQGSLQ.DMLQQLDLSPOC (SEQ. ID NO:35).

Date Recue/Date Received 2021-11-12 =
100591 Leptin A400: Leptin A400 is metreleptin with the serine residue at position 78 replaced with, a cysteine residue, as set forth following:
MVPIQKVQDDTKILIKTIVTRINDISFITQSVSSKQK.VTGLDFIPGLHPIL'FISK1vIDQTLAV

TEVVALSRLQGSLQDMLWQLDLSPGC (SEQ1 D NO: 36); to which a 20 kilodalton (kDa) PEG moiety has been attached via the cysteinc residue at position 78.
100601 Leptin MOO: Research by a number of investigators including the inventors has focused on the effects on aggregation or residue substitution in leptin. See e.g., Ricci et al., 2006. "Mutational approach to improve physical stability of protein therapeutics susceptible to . aggregation: Role of altered conformation in irreversible precipitation," Book. Chapter.
= MISBEHAVING PROTEINS: PROTEIN (MIS)FOLDING, AGGREGATION, AND STABILITY, Murphy .RM, Tsai AM, Eds., New York. Springer. pp. 331-350, Accordingly; leptin A560 with sequence following has been used in certain compounds and methods described herein:
MVPIQKVQDDIKTLIKTIVIRINDISHTQSVSSKQKVTGLEFIrGLHPILTLSKMDQTLAV

= = EVVALSRLQCSLQDMLQQLDLSPGC (SEQ ID NO:37).
100611 Leptin A100 Variants: Variants of Leptin A100 with the following amino acid substitutions follow:
D41E, H98S, W101Q, D109E, G1 13E, M1371, W139Q and G146E:
IvIVPIQKVQDDIKTLIKTIVIRINDISHTQSVSSKQKVTGLEFIPG LH PI LUSK IVI DQTLAv ..EVVALSRLQGSLQD1LQQLDLSPEC (SEQ ID NO: 38).
H98S, W101Q, A102T, G113E, M1371, W139Q, and C1146E:
MVPIQK.VQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAV

EVVALSRLQGSLQUILQQLDLSPEC (SEQ ID NO: 39).
H985, W101Q, Cill3E, M1371, WI39Q, and CH46E:

Date Recue/Date Received 2021-11-12 MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQISVTGLDFIPGLHPILTLSKMDQTLAV
YQQILTSMPSRNVIQISNDLIENLADLLIIVLAFSKSGSLPQASGLETLDSLGEVLEASGYST
EVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 40).
W101Q, G113E, M137I, W139Q, and G146E:
MVPIQKVQDDATLIKTEVTRINDISHIQSVSSKQKVTGLDFIPGLIIPILTLSKMDOILAV =
YQQ11,TSMPSRNVIQISNDLENIADUSEVLAFSKSCHLPQASGLETI,DSLGEVLEA.SGYST
EVVAISRLQGSLQDILQQIDLSPEC (SEQ ID NO: 41).
H98S, W101Q, M.1371, W139Q, and G146E:
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVIGLDFIPGLHPILTLSKMDQTLA V
YQQ1LTSMPSRNVIQISNDLENLRDLLEVLAFSKSCSLPQASGLETLDS1,GGVLEASGYST
EVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 42).
W101Q, G113E, M1371, W139Q,L143V, and G146E:
MVPIQKVQDDTICTLIKTIVTRINDISHIQSVSSKQKVIGLDFIPGLHPILTLSKMDQTLA
YQQ1LTSMPSRNVIQ1SNDLENUR.D.LLHVLA.FSKSCHLPQASGLETLDSLGEVIEASGYST
EVVALSRLQGSLQD1LQQLDVSPEC (SEQ ID NO: 43).
H98S, W101Q, A102T, M1371, W139Q, and G146E:
MVPIQKVQDDTKTLIKTIVIRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAV
YQQ1.1_,TSMPSRNVIQESNDLENIRDIA.HVLAFSK.SCSINTSGLETLDS1.:(1GVI,EASGYST
EVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 44).
H98S, W101Q, D109E, G113E, and G146E:
MVPIQKVQDDTICTLIK.TIVTR.INDISEINSVSSKOKVTGLDFIPGLI IPILMSKMDQTLAV

EVVALSRLQGSLQDMLwQLDLSPEC (SEQ ID NO: 45).
W101Q, M137I, W139Q, and GI46E:
MVPIQICVQDDTK.7.1KTIVTRINDISFITQSVSSKQKVTGLDFIPGUIPILTE,SKNIDQTLAV
= YQQ11-TSMPSRNVIQISNDLENLRDLLHVLAFSKSC1-ELPQASGLETLDSLGGVLEASGYS
TEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 46).

=
Date Recue/Date Received 2021-11-12 =
=
W101Q, M1371, W139Q, LI43V, and G146E
MVPIQKVQDDTICTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLA V
YQQ1LTSMPSRNVIQISNDLENLRDIA,HVLAFSKSCHLPQASGLETLDSLGGVLEASCAS
TEVVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO: 47).
H98S, W101Q, A102T, M1371, W139Q, L143V, and G146E:
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTOLDFIPGLHPILTLSKMDQTLAV
YQQ1LTSMPSRNVIQISNDLENLRDLLHVLA.FSK,SCSLPQTSGLETLDSLGGVLEASGYST =
EVVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO: 48).
H98S, W101Q, A102T, GI LIE, and G146E:
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAV

EVVALSRLQGSLQDMLWQLDLSPEC (SEQ II) NO: 49).
W101Q,G113E,andW139Q:
MVPIQKVQDDTKTLIKTIVTRINDISHIQSVSSKQKVTOLDFIPGLHPILTLSKMDQTLAV

EVVALSRLQGSLQDMLQQLDLSPGC (SEQ ID NO: 50).
W.101Q, Gl13E, W139Q, and G146E:
MVPIQK.VQDDIKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLIIPILILSKMDQTLAV
= YQQ1LTSMPSRNVIQISNDLENLRDLUTVLAFSKSCHIPQASGLETLDSLGEVLEASGYST
EVVALSRLQGSLQDMLQQLDLSPEC (SEQ 1D NO: 51).
chimeric Polypeptides 10062) In one aspect of the present disclosure, a series of chimeric polypcptides are described.
These chimeric polypeptides are based on a wild type seal leptin polypeptide wherein at least one contiguous region of 1-30 athino acids of a wild type seal leptin sequence has been replaced with a contiguous region of 1-30 amino acids of a mature human leptin sequence. A
wild type seal Date Recue/Date Received 2021-11-12 = , leptin sequence includes the sequence of wild type seal leptin (SEQ ID NO:28) and the sequence of wild type seal leptin with an N-terminal methionine (SEQ ID NO:31). A
mature human leptin sequence, useful for chimerizing wild type seal leptin as provided herein, includes the following sequences described above: mature human leptins (SEQ ID NO:12), mature human leptins with N-terminal methionine (SEQ ID NO:13), mature human leptin form 1 (SEQ ID
NO:20), mature human leptin form 2 (SEQ ID NO:21), mature human leptin form 3 (SEQ ID NO:22), mature human leptin form 4 (SEQ ID NO:23), mature human leptin form I with N-terminal methionine (Metreleptin, or A100, SEQ ID NO:24), mature human leptin form 2 with N-tenninal methionine (SEQ JD NO:25), mature human leptin form 3 with N-terminal methionine (SEQ ID
NO:26), mature human leptin form 4 with N-terminal methionine (SEQ ID .NO:27), A200 (SEQ ID
NO:34), A300 (SEQ ID NO:35), A400 (SEQ ID NO:36), A500 (SEQ ID NO:37), and variants (SEQ ID 'NO:38-51). In some embodiments, a series of chimeric pc lypeptides are described wherein at least one contiguous region of 1-30 arnino acids of a wild type seal leptin sequence (SEQ II) NO. 28 or SEQ ID NO:31) has been replaced with a contiguous region oft-30 amino acids of A100 (SEQ ID NO. 24).
[0063j In any of the disclosed chimeric polypeptides, a contiguous region of 1-30 amino acids can comprise any naturally or non-naturally occurring amino acid. Any combination of amino acids can be employed without restriction. 'That is, two or more amino acids in a contiguous region can be replaced with a naturally occurring amino acid, a non-naturally occurring amino acid, a conservative substitution, a non-conservative substitution or any combination thereof.
100641 The chimeric polypeptides described herein have demonstrated biological activity, in addition to enhanced physical properties. For example, the seal-human chimeric polypeptides show leptin activity in vitro and in vivo, The chimeric polypeptides also show enhanced stability and solubility compared to the mature human leptin polypeptides which are used to derive the sequences, as shown by the Examples.
[00651 The term "leptin activity" includes leptin binding activity and leptin functional activity.
The skilled artisan will recopize leptin analog compounds with leptin activity using suitable assays for measuring leptin binding or leptin functional activity. Leptin analog compounds can have an ICsa of about 200 nNI or less, about 100 nM or less, or about 50 nlvl or less. or about 5 nM or less, or about I iiM or less, in a leptin binding assay, such as that described herein The term "IC" refers in the customary sense to the half maximal inhibitory concentration of a =
Date Recue/Date Received 2021-11-12 compound inhibiting a biological or biochemical function. Accordingly, in the context of receptor binding studies, 1050 refers to the concentration of a test compound which competes half of a known ligand from a specified receptor. Leptin analog compounds can have an EC50 of about 20 nM or less, about 10 nM or less, about 5 nM or less, about I nM or less, or about 0.1 = nM or less, in a leptin functional assay, such as that described herein.
The term "EC,30" refers in the customary sense to the effective concentration of a compound which induces a response halfway between a baseline response and maximum response, as known in the art.
A. - Chimeric Polypeptides Incorporating Human Helix 1 [00661 The Helix I region of a mature human leptin polypeptide spans a contiguous region of 20 amino acids. Helix I and Helix 3 are antiparal lel helices that form part of Binding Site 11 of leptin to its receptor. This site interacts with the cytokine receptor homology domain (CR.1-1) of the leptin receptor and is thought to be a major receptor binding site, but not involved in receptor activation. See, for example, Peelman et al., 2004, J. Biol. Chem. 279: 41038.
100671 In one aspect, the present disclosure relates to chimeric polypeptides that arc based on .=
wild type seal leptin with an incorporated helix 1 sequence from mature human leptin. In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID .NO:28), wherein the contiguous region spanning the amino acids at positions 3-22 of SEQ ID NO:28 has been replaced with a contiguous region spanning the amino acids at positions 5-24 of A100 (SEQ ID NO:24). In some embodiments, a chimeric polypeptide comprises the sequence described in SEQ ID NO:52:
Seal teptiti with amino acids 3-22 replaced with amino acids 5-24 (helix 1) of metreleptin, respectively:
PIQKVQDDTXTLIKTIVTIUNDISPPQGVCSRPRVAGLDF1NVQSVRTLSGMDQII_ATYQ
QILTSLQSRSITVQ1ANDLANLRALLRLLASAKSCPVPRAR.GSDTIKGEGNVERASVHSTE
VVALSRLKAALODMLRQLDRN.PGC (SEQ ID Nft52).
[00681 In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine.(SEQ ID
NO:31), wherein the contiguous region spanning the amino acids at positions 3-22 of SEQ ID NO:31 has been =
Date Recue/Date Received 2021-11-12 replaced with a contiguous region spanning the amino acids at positions 5-24 of A100 (SEQ ID
NO:24).. In some embodiments, a chimeric polypeptide comprises the sequence described in.
SEQ ID NO:53:
Seal leptin with N7terminal methionine, and with amino acids 3-22 replaced with amino acids 5,24 (helix 1) of metreleptin, respectively:
MPIQKVQDDTKTLIKTIVIRINDISPPQGVCSRPRVAGI,DFIPR.VQSVRILSON1DQILATY

EVVALSI2LKAALQDNI.I.,RQLDRNPOC. (SEQ ID NO:53).
B. Chimeric Polypeptides Incorporating Human Helix 2 = 100691 The Helix 2 region of a mature human leptin poly-peptide spans a region of 16 contiguous amino acids. This helix is buried in the 4-helix bundle as described in the original crystal structure paper by Zhang et al. (Nature 1997 387:206).
10070] In one aspect, theipresent disclosure relates to chimeric. polypeptides that are based on wild type seal leptin with an incorporated helix 2 sequence from mature human leptin. In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide (SEQ lID NO:28), wherein the contiguous region spanning the amino acids at positions 50-65 of SEQ ID NO:28 has been replaced with a contiguous region spanning the - amino acids at positions 52-67 of A100 (SEQ ID NO:24). in some embodiments, a chimeric .polypeptide comprises the sequence described in SEQ ID NO:54:
Seal leptin with amino acids 50-65 replaced with amino acids 52-67 (helix 2) of rnetreleptin, respectively:
=PIQRVQDDTKTLIKTIITRJNDISPPQGVCSRPRVAGLDFIPRVQSVRTLSKM DQT LA V YQ
QILTSLQS RSVVQIANDLANLRALLRLLASAK.SCPVPRARGS DTI KGLGNVLRASVHSTE
VVALSRLKAALQDMLRQLDRNEGC (SEQ ID NO:54).
100711 in some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal Icptin polypcptide with an N-terminal methionine (SEQ ID
NO:31), wherein the = contiguous region spanning the amino acids at positions 50-65 of SEQ ID
NO:31 has been Date Recue/Date Received 2021-11-12 =
=
replaced with a contiguous region spanning the amino acids at positions 52-67 of A100 (SEQ ID
-N0:24). In some embodiments, a chimeric polypeptide comprises the sequence described in SEQ 1D NO:53:
Seal leptin with N-terminal methionine, and with amino acids 50-65 replaced with amino acids 52-67 (helix 2) of metreleptin, respectively:
MP 1.QRVQDDIKTLI KTIITRINDISPPQGVCS RPR.VAGLDFIPRVQS VRT1.8 K.MDQT LA VY

EVVAL,SRLKAALQDMI,RQI.,DRNPGC (SEQ ID NO:55), C. Chimeric Polypeptides incorporating Human Helix 3 = (00721 The Helix 3 region of a mature human leptin polypeptide spans a contiguous region of 22 amino acids. Helix 3 and Helix I are antiparallel helices that form part of Binding Site 11 of leptin to its receptor. This site interacts with the cytokine receptor homology domain (CRH) of the leptin receptor and is thought to he a major receptor binding site, but not involved in receptor activation. Sec, for example, Peelman et al., 2004, J. Biol. Chem. 279: 4103g, =
100731 In one aspect, the present disclosure relates to chimeric polypeptides that are based on A
wild type seal leptin with an incorporated helix 3 sequence from mature human leptin. In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild -type seal leptin polypeptide (SEQ ID NO:28), wherein the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:28 has been replaced with a contiguous region spanning the .
amino acids at positions 73-94 of A100 (SEQ ID NO:24). In some embodiments, a chimeric poly-peptide comprises the sequence described in SEQ ID-NO:29:
Seal leptin with amino acids 71-92 replaced with amino acids 73-94 (helix 3) of tnetreleptin, respectively:
PIQRVQDDTKTLIK.IIITRINDISPPQGVCSRPRVAGEDFJPRVQSVRTLSOM DQ11..ATYQQ
ILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVV
A LSRLK.AA.1..QDMLRQ1,,DRNPOC (SEQ I.D NO:29).

Date Recue/Date Received 2021-11-12 100741 In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID
NO:31), wherein the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:'31 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A100 (SEQ ID
NO:24). In some embodiments, a chimeric polypeptide comprises the sequence described in SEQ 1D NO:32:
Seal leptin with N-terminal methionine, and with amino acids 71-92 replaced with amino acids 73-94 (helix 3) of metreleptin, respectively:
MPIQRVQDDIKTLIKIIITIZINDISPPQGVCSIZPRVACiLDFIPRVQSVRTLSGMDQILATYQ
QILTSLQSRNVIQISNDLENLRDLIHVLAFSKSCPVPRARGSDIIKGLGNVLRASVHSTEV
VALSRLK_AALQDMLRQLDRNPGC (SEQ I.D.N0:32).
D. Chimeric Polypeptides Incorporating Human Helix 4 100751 The Helix 4 region of a mature human leptin polypeptide spans a contiguous region of 22 amino acids. Helix 4 is thought to form parts of Binding Site 1 and Binding Site Ill of leptin, both of which are important for receptor activation. See, for example, Peelman et al., 2004, J.
Biol. Chem. 279: 41038. . 6 =
100761 In one aspect, the present disclosure relates to chimeric polypeptides that are based on wild type seal leptin with an incorporated helix 4 sequence from mature human leptin. In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type scat leptin polypeptide (SEQ ID NO:28), wherein the contiguous region spanning the amino acids at positions 120-141 of SEQ ID NO:28 has been replaced with a contiguous region spanning the amino acids at positions 122-143 of A100 (SEQ ID NO:24). In some embodiments, a chimeric polypeptide comprises the sequence described in SEQ ID NO:56:
Seal leptin with arnintracids 120-141 replaced with amino adds 122-143 (helix 4) of metreleptin, respectively:
PIQR,VQDDTKT1.1 KTI IT RI N DI SPPQGVC.SRP R.V A GLDFIPRVQSVRTI..SGMDQII.A
TYQQ
Date Recue/Date Received 2021-11-12 =

vALSRLQGSLQDMLIVOLD.I.,NPOC (SEQ ID NO:56).
=
100771 In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID
NO:31), wherein the contiguous region spanning the amino acids at positions 120-141 of SEQ ID
NO:31 has been replaced with a contiguous region spanning the amino acids at positions 122-143 of A100 (SEQ
ID NO:24). In some embodiments, a chimeric. polypeptide comprises the sequence described in SEQ ID NO:57:
Seal leptin with N-terminal methionine, and with amino acids 120-141 replaced with amino acids 122-143 (helix 4) of metreleptin, respectively:
WIQRVQDDIKTLIKTUTRINDISPPQGVCSRPRVAOLDFIPRVQSVRTLSGMDQILATYQ

VVALSILLQGSLQDML,WQLDLNPOC (SEQ ID NO:57).
E. Chimeric Polypeptides Incorporating Human AB Loop 100781 'rhe AB Loop legion of a mature human leptin polypcptide spans.a contiguous r'egion of 27 amino acids. The All Loop is thought to form part of Binding Site III as well as a small portion of Binding Site 1 of leptin. See, for example, Peelman et al.? 2004,1 Biol. Chem. 279:
41038. This region also contains the absolutely conserved motif CIDEIP (SEQ ID
NO: 164).
100791 In one aspect, the present disclosure relates to chimeric poiypeptides that are based on wild type seal leptin with an incorporated AB Loop sequence from mature human leptin. In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:28), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:28 has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A1.00 (SEQ ID NO:24). In some embodiments, a chimeric poly-peptide comprises the sequence described in SEQ ID NO:58:

Date Recue/Date Received 2021-11-12 Seal leptin with amino acids 23-49 replaced with amino acids 25-51 (AB loop) of metreleptin, respectively:
PIQRVQDDTKTLIKTIIITRINDISHTQSVSSK.QKVTGLDEIPOLITPILTLSOMDQILATYQQ1 A LSRLICAALQDMLRQLDRNPOC (SEQ ID 'NO:58).
100801 In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal inethionine (SEQ ID
NO:31), wherein the contiguous region spanning the amino acids at positions 23-49 or SEQ ID NO:31 has been replaced with a contiguous region spanning the. amino acids at positions 25-51 of A100 (SEQ ID
NO:24). In some embodiments, a chimeric polypeptide comprises the sequence described in -SEQ ID NO:59:
Seal leptin with N-terminal methionine, and with amino acids 23-49 replaced .with amino acids 2.5-51 (AB loop) of metreleptin, respectively:
MP1Q.R.VQDDT.KTLIKTIITRINDISHTQSVSSKQKVTGLDEI.PGLIIPILTLSGMDQILATYQ
QELTSLQSRSVVQ1ANDLANLKALLICLASAKSCPVPRARGSDTIMUGNVLIZASVHsTE
ATVALSRLICAALQDMLRQI.DRNPOC (SEQ ID NO:59).
F. Chimeric Polypeptides Incorporating Human Loop 3-4 100811 The Loop 3-4 region of a mature human leptin polypeptide spans a contiguous region of 27 ammo acids. Loop 3-4 is thought to contain a part of Binding Site III of leptin to its receptor.
Sec, for example, Peelman et al., 2004, .1. Biol. Chem, 279: 41038.
100821 In one aspect, the present disclosure rotates to chimeric polypeptides that Arc based on wild type seal leptin with an incorporated Loop 3-4 sequence from mature human leptin. In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:28), wherein the contiguous region spanning the amino acids at positions 93-119 of SEQ ID NO:28 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of A100 (SEQ ID NO:24). In some embodiments, a chimeric polypeptide comprises the sequence described in SEQ ID NO:60:

Date Recue/Date Received 2021-11-12 Seal leptin with amino acids 93-119 replaced with amino acids 95-121.(loop 3-4) of inetreleptin, respectively:
PIQRVQDDIKTLIKTIITRINDISPPOGVCSRPRVAGLDEIPRVOSVRTLSGMDQILATYQQ
ILTSLOSRSVVQ1ANDLANLRALLRLLASAKSCHLPWASCiLETLDSLGGVLEASOYS1EV
VALSRLKAALQDMLROLDRNPOC (SEQ ID NO:60).
100831 En some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID
NO:31), wherein the contiguous region spanning the amino acids at positions 93-119 of SEQ ID NO:3 I has been replaced with a contiguous region spanning the amino acids at positions-95-121 of A100 (SEQ
ID NO:24). In some embodiments, a chimeric polypeptide comprises the sequence described in SEQ ID NO:61:
Seal leptin with N-terminal methionine, and with amino acids 93-119 replaced with amino acids 95-121 (loop 3-4) of met releptin, respectively:

QIUTSLQSRSVVQ1ANDLANLRALLIZ.LLASAKSC111..PWASOLETLDS1..GGVLEASGYSTE
VVALSRLKAALQDMLROLDRNI)GC (SEQ ID NO:61).
G. Chimeric Combination Polypeptides 100841 In another aspect of the Present disclosure, a series of chimeric combination polypeptides are described. These chimeric combination polypeptidcs arc based on a wild type' seal leptin polypeptide wherein two or more contiguous regions of 1-30 amino acids of a wild type seal leptin sequence (for example, SEQ ID NO:28 or SEQ ID NO:31).have been replaced at each region with a contiguous region of.I-30 amino acids of a mature human leptin sequence.
Chimeric combination polypeptidcs can be engineered to demonstrate enhanced physical properties compared to the mature human leptin polypeptides which are used to derive the sequences, while retaining the biological activity of human leptin.
100851 In some embodiments, the present disclosure relates to chimeric polypeptides that are based on wild type seal leptin with an incorporated helix 1 sequence and an incorporated helix 3 = 23 Date Recue/Date Received 2021-11-12 >
sequence from mature human leptin. In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin pulypeptide .(SEQ ID
NO:28), wherein the contiguous region spanning the amino acids at positions 3-22 of SEC) 113 .N0:28 has been replaced with a contiguous region spanning the amino acids at positions 5-24 of A100 (SEQ ID
NO:24); and the contiguous region spanning the amino acids at positions 71-92 of SEQ ID
NO:28 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A100 (SEQ ID NO:24). In some embodiments, a chimeric polypeptide comprises the = sequence described in SEQ ID NO:62:
Seal leptin with amino acids 3-22 replaced with amino acids 5-24 (helix 1) of metreleptin, and amino acids 71-92 replaced with amino acids 73-94 (helix 3) of metreleptin, respectively:
PIQKVQDDTKTLIKTIVTRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQ
QILTSLQSRNVIQIISNDLENLRDLLHVLAFSKSCPVPRARGSDTEKOLONVLKASVIISTEV
VALSIZLKAALQDMLIZQLDRNPOC (SEQ ID NO:(i2) 100861 In some embodiments, a chimeric polypeptide comprises the amino acid siquencc of a =
wild type seal leptin poly-paptidc with an N-terminal methionine (SEQ ID
NO:31), wherein the 'contiguous region spanning the amino acids at positions 3-22 of SEQ ID NO:31 has been replaced with a contiguous region spanning the amino acids at positions 5-24 of A100 (SEQ ID
.NO:24), and the contiguous region spanning the amino acids at positions 71-92 of SEQ ID
NO:31 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A.100 (SEQ ID NO:24). In sonic embodiments, a chimeric polyp:vide comprises the sequence described in SEQ ID NO:63:
= Seal leptin with IN-terminal methionine, and with amino acids 3-22 replaced with amino acids 3-24 (helix I) of metreleptin, and amino acids 72-93 replaced with amino acids 73-94 (helix 3) of metreleptin, respectively:

WALSRLICAALQD1vILRQLDRNPGC (SEQ ID NO.63) .
.
Date Recue/Date Received 2021-11-12 =
100871 In some embodiments, the present disclosure relates to chimeric polypeptides that are based on wild type seal leptin with an incorporated helix 3 sequence and an incorporated AB
Loop sequence from mature human leptin. In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide (SEQ.) DNO:28), wherein the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:28 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A.100 (SEQ ID NO:24), and the contiguous region spanning the amino acids at positions 23-49 of SEQ
ID NO:28 has been replaced with a contiguous region spanning the amino acids at positions 25-.
51 of A100 (SEQ ID NO:24). In some embodiments, a chimeric. poly-peptide comprises the sequence described in SEQ ID NO:64:
Seal leptin with amino acids 71-92 replaced with amino acids 73-94 (helix 3) of rnetreleptin, and with amino acids 23-49 replaced with amino acids 25-51 (AB loop) of metreleptin, respectively:
PIQRVQDDTE.TLIK.T.IITRINDISIITQSVSSK.QKVTGLDPIPG1.11.PILTLSCits4DQILAIXQQ1 LTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVV
ALSRLKAALQDNILRQEDIINPCIC (SEQ ID NO:64) [00881 In some embodiments, a chimeric poly-peptide comprises the amino acid sequence of a wild type seal leptin polypeptide with an N-tennina I methionine (SEQ ID
NO:31), wherein the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:31 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A100 (SEQ ID
NO:24), and the contiguous region spanning the amino acids at positions 23-49 of SEQ ID
.NO:31 has been replaced with a contiguous region spanning the amino acids atpositions 25-51 of A100 (SEQ ID NO:24). In some embodiments, a chimeric polypeptide comprises the sequence described in SEQ ID NO:65:
Seal leptin with N-terminal methionine, and with amino acids 71-92 replaced with amino acids 73-94 (helix 3) of metreleptin, and with amino acids 23-49 replaced with amino acids 25-51 (AB loop) of metreleptin, respectively:
Date Recue/Date Received 2021-11-12 MPIQRVQDDTKTLIKTTITRINDISHTQSVSSKQKVTGLDFIPOLHPI LTLSGMDQILATYQ
QILTSLQSRNIIIQISNDLENI,RDLLFIVLAFSKSCPVPRARGSDTIKGLONVLRASVHSTEV
.VALSRLICAALQDMLRQLDRNPOC (SEQ ID NO:65) 1011891 In some embodiments, the present disclosure relates to chimeric polypeptides that are based on wild type seal leptin with an incorporated helix 3 sequence and an incorporated Loop 3-4 sequence from mature human leptin. In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:28), Wherein the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:28 has been replaced with a Contiguous region spanning the amino acids at positions 73-94 oil\ 100 (SEQ IL) NO:24), and the contiguous region spanning the amino acids at positions 93-11
9 or SEQ ID
-N0:28 has been replaced with a contiguous region spanning the amino acids at positions 95-121 =
of A100 (SEQ ID NO:24). In sonic embodiments, a chimeric polypeptide comprises the sequence described in SEQ ID NO:66:
Seal leptin with amino acids 71-92 replaced with amino acids 73-94 (helix 3) of metreleptin, .
and with amino acids 93-119 replaced with amino acids 95-121 (loop 3-4) of metreleptin.
respectively:
PIQRVQDDTKIIIKTIERINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSOMDQILATYQQ
ILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEW =
ALSRLKAALQDMILR.QLDRNPOC (SEQ ID NO:66).
100901 In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a = wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID
NO:31), wherein the contiguous region spanning the amino acids at positions 71-92.of SEQ ID NO:31 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A100 (SEQ ID
NO:24), and the contiguous region spanning the amino acids at positions 93-119 of SEQ ID
NO:31 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of MOO (SEQ ID NO:24). In some embodiments, a chimeric polypeptide comprises the sequence described in SEQ ilD NO:67:
=
.26 Date Recue/Date Received 2021-11-12 Seal leptin with N-terminal methionine, with amino acids 71-92 replaced with amino acids 73-94 (helix 3) of metreleptin, and with amino acids 93-119 replaced with amino acids 95-12.1 (loop 3-4) of metreleptin, respectively:
MPIQRVQDDTKTLIKTEITRINDISPPQGVCSRPRVAGLDFIPRINSVRTLSGMDQILATYQ
QILTSLQSRNVIQISNDLENLRDWiVLAYSKSCHLI'WASGLETLDSLGGVLEASGYsTEv VALSRLKAA LQDMLRQLDRNPGC (SEQ ID NO:67) 100911 In some embodiments, the present disclosure relates to chimeric polypeptides that are based on wild type seal leptin with an incorporated AB Loop sequence and an incorporated helix 4 sequence from mature human leptin. In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type scat leptin polypcptide (SE() ID
NO:28), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:28 has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A100 (SEQ ID
NO:24), and the contiguous region spanning the amino acids at positions .120-141 of SEQ ID
NO:28 has been replaced with a contiguous region spanning the amino acids at positions 122-143 of A 100 (SEQ ID NO:24). In some embodiments, a chimeric polypeptide comprises the sequence described in SEQ 'NO:68:
Seal leptin with amino acids 23-49 replaced with amino acids 25-51 (A13 loop) of metreleptin, and with amino acids 120-141 replaced with amino acids 122-143 (helix 4) of metreleptin, respectively:
PIQRVQDDTKILIKTIITRINDISHTQSVSSKQKVTGLDFIPOLHI'ILTLSGMDQILATYQQ1 ALSRLQGSLQDMLWQLDENPGC (SEQ ID NO:68) (00921 In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ 10 NO:31), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:31.
has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A100 (SEQ ID
'NO:24), and the contiguous region spanning the amino acids at positions 120-141 of SEQ ID
NO:31 has been replaced with a. contiguous region spanning the amino acids at positions 122-.27 =
Date Recue/Date Received 2021-11-12 =
143 of A MO (SEQ ID NO:24). In some embodiments, a chimeric polypeptide comprises the 'sequence described in SEQ ID NO:69:
Seal leptin with N-terminal methionine, with amino acids 23-49 replaced with amino acids 25-51 (AR loop) Of metreleptin, and with amino acids 120-141 replaced with amino acids 122-143 (helix 4) of Metreleptin, respectively:
MPI QR.VQDDTKILIKTI1TRIN DIS FITQS V SS KQK VTGLDFIPGLIIPILTISGMDQ I LATYQ
QILTSLQSRSVVQIANDLANLRALLRLL A SAKSCPVP RA ROSDTIKOLGNVLRASVHSTE
VVALSRLQGSLQDMI.WQLDLNP(iC. (SEQ ID NO:69) 100931 In some embodiments, the present disclosure relates to chimeric polypeptides that are based on wild type seal leptin with an incorporated AR Loop sequence and an incorporated Loop 3-4 sequence from mature human leptin. In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypcptide (SEQ
ID NO:28), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:28 . has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A100 (SEQ ID NO:24), and the contiguous region spanning the amino acids at positions 93-119 of SEQ ID NO:28 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of A100 (SEQ ID NO:24). In some embodiments, a chimeric polypeptide comprises ;he sequence described in SEQ NO:70:
Seal leptin with amino acids 23-49 replaced with amino acids 25-51 (AB loop) of metreleptin, and with amino acids 93-119 replaced with amino acids 95-121 (loop 3-4) of naetreleptin, respectively:
RIQRVQDDTKILIKTIITRINDISFITQSVSSKQKVTGLDFIPGIA-IPILTL.SGMDQ11.,A.TYQQ1 LTSLQSRSVVQ[ANDLANLRALLRLLASAKSCHLPWASGLEFLUSLOGVLEASGYSTEV
VALSRLKAALQDMI..RQLORNPGC (SEQ ID NO:70) 100941 In some embodiments, a chimeric polypeptidc comprises the amino acid sequence of a wild type seal leptin poly-peptide with an N-terminal methionine (SEQ ID
NO:31), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:31 has been Date Recue/Date Received 2021-11-12 replaced with a contiguous region spanning the amino acids at positions 25-51 of A100 (SEQ ID
NO:24), and the contiguous region spanning the amino acids at positions 93-119 of SEQ ID
.NO:31 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of A100 (SEQ ID NO:24). In some embodiments, a chimeric polypeptide comprises the sequence described in SEQ ID NO:71:
Seal leptin with N-terminal methiunine, with amino acids 23-49 replaced with amino acids 25-51 (AB loop) of metreleptin, and with amino acids 93-119 replaced with amino acids 95-121 (loop 3-4) of metreleptin, respectively:
MPIQRVQDDTKTLIKTITTRINDISIITQSVSSKQKYT(JLDFIPOLHPILTLSOMDQ1LATYQ
= QILTSLQSRSVVQIANDLANLRALLRLLASAKSCHLPWASOLETLDSLGGVLEASGYSTE
VVA L,S12.1.,K AA LQDM LRQLDRNEGC (SEQ ID NO:71).
100951 In some embodiments, the present disclosure relates to chimeric polypeptides that are based on wild type seal leptin with an incorporated AB Loop sequence, an incorporated .Loop 3-4 sequence, and an incorporated helix 3 sequence .from mature.human leptin. In some embodiments, a chimeric polypeptide comprises the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:28), wherein the contiguous region spanning the amino acids at, positions 23-49 of SEQ ID NO:28 has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A100 (SEQ ID NO:24), the contiguous region spanning the amino acids at positions 93-119 of SEQ ID NO:28 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of A100 (SEQ ID NO:24), and the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:28 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A100 (SEQ ID
NO:24). In - sonic embodiments, a chimeric polypeptide comprises the sequence described in SEQ ID NO:72:
Seal leptin with amino acids 23-49 replaced with amino acids 25-51 (AB loop) of metreleptin, with amino acids 93-119 replaced with amino acids 95-121 (loop 3-4) of metreleptin, and with amino acids 71-92 replaced with amino acids 73-94 (helix 3) of metreleptin, respectively:

Date Recue/Date Received 2021-11-12 LISEQSRNVIQISNDLENERDILFIVEAFSKSCHLPWASGLETEDSLOGVLEASGYSTEVII
.ALSRLKAALQDMERQLDRNPOC (SEQ ID NO:72) INN In some embodiments, a chimeric polypeptide comprises the amino acid.
sequence of a = wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID
NO:31), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:31 has beers replaced with a contiguous region spanning-thc amino acids at positions 25-51 of A100 (SEQ ID
NO:24), the. contiguous region spanning the amino acids at positions 93-1 19 of SEQ ID NO:31 ' has been replaced with a contiguous region spanning the amino acids at positions 95-121 of =
MOO (SEQ ID NO:24), and the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:28 has been replaced with a contiguous region spanning the amino acids at .positions 73-94 of A100 (SEQ ID NO:24). In some embodiments, a chimeric polypeptide comprises the sequence described in SEQ ID NO:73:
Seal,leptin with N-terminal methionine, with amino acids 23-49 replaced with amino acids 25-51 (AB loop) of metreleptin, with amino acids 93-119 replaced with amino acids 95-121 (loop 3-4) of metreleptin, and with amino acids 71-92 replaced with amino acids 73-94 j (helix 3) of metreleptin, respectively:
MPIQRVQDDTKTLIKTITTRINDISHIQSVSSKQKVTGLDFIPOLHPILTLSGMDQILATYQ -QILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEV
VALSRLKAALQDNILRQLDRNPGC (SEQ ID NO:73).
[00971 In some embodiments, the chimeric polypeptides provided by the invention contain a Cys to Scr amino acid substitution at position 30 or the wild type seal polyp:1)6de sentiency.
According to some embodiments, the following chimeric polypeptides are provided: "
Seal leptin with amino acids 30 and 3-22 replaced with amino acids 32 and 5-24 (helix 1) of metreleptin, respectively:
PIQKVQDDTKTLIKTIVTRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQ

VVALSRL1CAALQDMLRQLDRNPGC (SEQ ID NO:74).
Date Recue/Date Received 2021-11-12 Seal Ieptin with N-terminal methionine, and with amino acids 30 and 3-22 replaced with amino acids 32 and 5-24 (helix 1) of metreleptin, respectively:
MPIQKVQDDTKILIKTIVTRINDISPPQOVSSRPRVAGLDFIPRVQSVRTLSGMDQILATY
QQILTSLQSRSVVQIANDLANLRALLRLLASAKSCPVPRARGSDTIKGLONVLRASVHST
EVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:75).
Seal leptin with amino acids 30 and 50-65. replaced with amino acids 32 and 52-67 (helix 2) of metreleptin, respectively:
PIQIIVQDDTKTLIKIIITRINDISPPQGVSSRPRVAGLDHPRVQSVRTLSKVIDQTLAVYQQ

VALSRLKAALQDMLRQLDRNPOC (SEQ ID NO:76).
Seal leptin with N-terminal methionine, and with amino acids 30 and 50-65 replaced with amino acids 32, and 52-67 (helix 2) of metreleptin, respectively:
MPIQRVQDDTKTLIKTIITRINDISPPQGVSSR.PRVAGLDF.IPRVQSVRTLSKMDQTLAVY

EVVALSRLKAALQDMLRQLDRNPOC(SEQ ID NO:77).
Seal leptin with amino acids 30 and 71-92 replaced with amino acids 32 and 73-94 (helix 3) of metreleptin, respectively:
PIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSOMDQILATYQQ I
LTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKOLONVLRASVHSTEVV
ALSRLKAALQDMLRQLDRNPGC (SEQ ID NO :30).
Seal leptin with N-terminal methionine, and with amino acids 30 and 71-92 replaced with amino acids 32 and 73-94 (helix 3) of metreleptin, respectively:

VALSRLKAAIQDIALRQL.DRNPOC (SEQ 1D'NO:33).

Date Recue/Date Received 2021-11-12 =
Seal leptin with amino acids 30 and 120-141 replaced with amino acids 32 and (helix 4) of metreleptin, respectively:
PIQRVQD.DTKI1IKIIITRINDISPPQGVSSRPRVAGLDF1PRVQSVRTLSOMDQILATYQQ1 ALSRLQGSLQDML,WQLDLNPGC (SEQ ID NO:78).
Seal leptin with N-terminal rnethionine, and with amino acids 30 and 120-141 replaced with amino acids 32 and 122-143 (helix 4) of metreleptin, respectively:
M.PIQRVQDDTKILIKTI1TRINDISPPQGVSSRPR.VACiLDFIPRVQSVIM.SGMDQILATYQ
QILTSLQSRSVVQ1ANDLANLKALLRLLAS.AKSCPVPIZARGSDTIKOLONVLItASVHSTE
VVALSRLQGSLQDMEWQLDLNPGC (SEQ ID NO:79).
Seal leptin with amino acids 30 and 93-119 replaced with amino acids 32 and 95-121 (loop 3-4) of metreleptin, respectively:
= PIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSOMDQILATYQQ1 LTSEQSRSINQIANDLANLRALIALLASAKSCHLPWASGLEILDSLCOVI,EASGYSTEV
VALSRLKAALQDMIAQLDRNPGC (SEQ ID NO:80).
Seal leptin with N-terminal methionine, and with amino acids 30 and 93-119 replaced with .
amino acids 32 and 95-121 (loop 3-4) of metreleptin, respectively:
NIPIQRVQDDIKTLIKTIITRINDISPNOVSSRPRVAGLDEIPRVQSVRILSGMDQILATYQ

VVALSRLKAALQDMERQLDRNPOC (SEQ ID NO:8 I).
Seal leptin with amino acid 30 replaced with amino acid 32, amino acids 3-22 replaced with amino acids 5-24 (helix I) of metreleptin, and amino acids 71-92 replaced with amino acids 73-94 (helix 3) of metreleptin, respectively:
PIQKVQDDTKILIKTIVIIIINDISPNOVSSRPIWAOLDEIPKVQSVR.ILSOMDQILATYQ
QILTSLQSRNVIQISNDLENLIZDELHVIAFSKSCPVPRARGSDTIKGEGNVLRASVHSTEV
VALSRLKAALQDMLRQLDRNPOC (SEQ ID .N0:82) Date Recue/Date Received 2021-11-12 Seal leptin with N-terminal methionine, and with amino acid 30 replaced with amino add 32, amino acids 3-22 replaced with amino acids 5-24 (helix 1) of metreleptin, and amino acids 72-93 replaced with amino acids 73-94 (helix 3) of metreleptin, respectively:
MPIQKVQDDIKTLIKTIVTRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSOMDQILATY
QQI.LTSLQSRNVIQISNDLENLIIDLLHVLAFSKSGPVPRARGSDTIKOLGNVLRASVHSTE
VVALSRLICAALQDMLRQLDRNPGC (SEQ ID NO:83) Seal leptin with amino acid 311 replaced with amino acid 32, amino acids 71-92 replaced with amino acids 73-94 (helix 3) of metreleptin, and with amino acids 93-119 replaced with amino acids 95-121 (loop 3-4) of metreleptin, respectively:

LTSLQSRNVIQISNDLENLRDLEHVLAFSKSCHLPWASOLETLDSLGGVLEASGYSTENIV
ALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:84) Seal leptin with N-terminal methionine, with amino acid 30 replaced with amino add 32, amino acids 71-92 replaced with amino acids 73-94 (helix 3) of metreleptin, and with amino acids 93-119 replaced with amino acids 95-121 (loop 3-4) of metreleptin, respectively:
- MPIQRVQD.DTKTLIKT1.1TR.INDISPPQCIVSSRPRVAGLDFEPRVQSVRTLSOMDQILATYQ
QILTSLQSRNVIWNDLENLRDLLHVLAESKSCHLPWASGLETLDSLOCVLEASGYSTEV
VALSRLICAALQDMLRQLDRNPGC (SEQ ID NO:85).
=
= [00981 Further embodiments. It is understood that each of the polypeptidcs disclosed herein are also contemplated to include (optionally) a inethioninc at the N-terminus in frame with the naturally-occurring first amino acid thereof. For example, nictretcptin (leptin A100) consists of mature hurnan leptin to which has been added an N-terminal methionine, as disclosed in SEQ ID
NO:24. Similarly, a methionine residue may be included at the.N-terminus of any of the amino acid sequences and Formulae disclosed herein throughout.
100991 in some embodiments, chimeric polypeptide analogs are provided. A
chimeric polypeplide analog can have at least 80%, for example 80%, 85%, 90%, 95%, 98%
or even higher, sequence identity relative to a parent chimeric poly-peptide. in some embodiments, the parent chimeric polypeptide is a polypeptide set out in SEQ ID NO:29, SEQ ID
NO:30, SEQ ID

= =
Date Recue/Date Received 2021-11-12 = P
NO:32, SEQ ID NO:33, SEQ ID NO:52, SEQ ID NO:53, SEQ ID N0:54, SEQ ID NO:55, SEQ
ID NO:56, SEQ ID NO:57, SEQ LD NO:58, SEQ ID .N0:59, SEQ ID NO:60, SEQ ID
NO:61, SEQ ID NO:62, SEQ SEQ D NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ
NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ
ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID
NO:78, SEQ ID NO:79, SEQ ID NO:80, ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID
NO:84, or SEQ ID NO:85. Accordingly, in some embodiments, a chimeric polypeptide analog may have at least 80%, for example 80%, 85%, 90%, 95%, 98% or even higher, sequence identity relative to .
any chimeric polypeptide selected from the group consisting of st-1Q ID NO:29, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID INO:33. In some embodiments, a chimeric polypeptide analog may have at least 80%, for example 80%, 85%, 90%, 95%, 98% or even higher, sequence identity relative to the chimeric -polypeptide set forth in SEQ ID NO:33. In some embodiments, a = chimeric polypeplide analog may have at least 90% sequence identity relative to the a chimeric polypeptide set forth in SEQ ID NO:29,-SEQ ID NO:30, SEQ 1D NO:32, or SEQ ID
NO:33. In some embodiments, a chimeric polypeptide analog may have at least 90% sequence identity relative to the chimeric polypeptide set forth in SEQ ID NO:33.
[0.1001 Additionally, chimeric polypeptide analogs may he designed, prepared.
and used in accordance with the invention in which 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, IS, 19, 20 or even 21 amino acids Fa chimeric polypeptide selected from the group consisting of:
SEQ ID NO:29, SEQ 1D NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:52, SEQ ID
.N0:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ
ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID .N0:63, SEQ ID
NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:?], SEQ ID .N0:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID .N0:75, SEQ
ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, ID NO:81, SEQ ID
NO:82, SEQ ID NO:83, SEQ ID NO:84, and SEQ ID NO:85, is/are substituted with another amino acid, such as a conservative. amino acid or a non-conservative amino acid, or is/are otherwise altered. As custo.mary in the art, the term "conservative" in the context of amino acid substitutions refers to substitution which maintains properties of charge type (e.g., anionic, cationic, neutral, polar and the like), hydrophobicity or hydrophdicity, bulk (e.g., van der Waals = 34 =
Date Reeue/Date Received 2021-11-12 contacts and the like), and/or functionality (e.g., hydroxy, amine, sulhydryl and the like). The term "non-conservative" refers to an amino acid substitution which is not conservative.
101011 In another aspect, the invention provides chimeric polypeptide analogs comprising at least one contiguous region of 1-30 amino acids front a mature human leptin analog sequence that contains at least one amino acid substitution at a position where divergence is observed in a corresponding position in a leptin from another species.
101021 As is understood in the art, for example, murine leptins, rat leptins, bovine leptins, porcine leptins, and rhesus monkey leptins, such as those disclosed herein, are each substantially homologous to human leptins; in particular, the mature forms of these leptins are substantially homologous to mature leptins, and further, particularly near the N-terminal portion of the protein. One may prepare analogs of such leptins, such as mature human leptin form 1 (SEQ .11) NO:20) and metrcleptin (SEQ ID NO:24), such as by substituting or otherwise altering amino acid residues at one or more positions in such sequences where divergence is observed in a corresponding mature mouse, rat, bovine, porcine, or rhesus monkey leptin. For example, mature human leptins (e.g., SEQ 1.11) NO:20) elicits biological responses in, for example, mice, rat, and monkey). See, e.g., WO 98/28427, WO 2009/064298, US2007/0020284, US2008/0207512,and Murakami et al., 1995, Biochem. Biophys. Res. Comm. 209:
944-952.
Because human mature leptins have biological activity in, e.g., such specie's, leptin analogs may be designed and prepared in which one or more amino acids at positions which arc divergent at the corresponding position(s) in a leptin from one or more of such species are substituted with the amino acid(s) at such corresponding divergent positions.
101031 For example, using a human mature leptin protein according to SEQ ID
NO:20 wherein the first amino acid is valine and the amino acid at position 146 is cysteine, one may substitute with another amino acid one or more of the amino acids at positions 32, 35, 50, 64.68, 71, 74, 77, 89, 97, 100, 101, 105, .106, 107, 108, 111, 112, 118, 136, 138, 142, and 145 with the corresponding amino acid(s) found at the corresponding position(s) in SEQ ID
NO:2 in order to design and prepare leptin analogs comprised by the chimeric polypeptides in accordance with the invention. Additionally, one may also substitute another amino acid, such as a conservative amino acid or a non-conservative amino acid, into one or more of positions 32, 35, 50,64, 68, 71, 74, 77, 89, 97, 100, 101, 105, 106, 107, 108, Ill, 112, 118, 136, 138, 142, and 145 or, for =
Date Recue/Date Received 2021-11-12 example, SEQ ID NO:20 in order to design and prepare leptin analogs comprised by the chirnenc polypeptides in accordance with the invention.
[01.041 One may further prepare additional leptin analogs based on the mature rat leptin protein sequence (SEQ ID NO:16). See, e.g., WO 98/28427, VS2007/0020284, and Murakami etal., 1995, id. Mature rat leptin differs from mature human leptin form 1 (SEQ ID 'NO:20) at the following positions: 4, 32, 33, 35, 50, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138 and 145. Accordingly, at one or more of such positions in SEQ ID NO:20, one may substitute the amino acid found at the corresponding position(s) found in mature rat leptin (SEQ
ID NO:16) in order to design and prepare leptin analogs comprised by the chimeric polypeptides in accordance with the invention. Additionally, one may also substitute another amino acid, such as a conservative amin.o acid or a non-consetvative amino acid, into one or more of positions 4, 32, 33, 35, 50, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138 and 145 of, for example, SEQ ID NO:20, in order to design and prepare leptin analogs comprised by the chimeric polypeptides in accordance with the invention 101051 The positions from both mature rat leptin (SEQ ID NO:16) and mature murine leptin form I (SEQ ID NO:2) which diverge from the mature human leptin form 1 (SEQ ID
NO:20) are: 4, 32, 33, 35, 50,64, 68, 71, 74, 77, 18, 89,97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145. Accordingly, at one or more of such positions in SEQ
ID NO:20, one may substitute the amino acid found at the corresponding position(s) found in mature rat leptin sequence. (SEQ ID NO:16) or mature marine form 1 sequence (SEQ ID NO:2) in order to design and prepare leptin analogs comprised by the chinieric polypeptides in accordance with the invention, Additionally, one may also substitute another amino acid, such as a conservative amino acid or a non-conservative amino acid, into one or more of positions 4, 32, 33, 35, 50, 64, 68,71, 74, 77, 78, 89, 97, IOU, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145 in order to design and prepare leptin analogs comprised by the chimeric polypeptides in accordance with the invention.
101061 In addition, the amino acids found in rhesus monkey mature leptin (SEQ
ID NO:14) which diverge from mature human leptin forrn 1 (SEQ ID NO:20) are (with amino acid residues noted in parentheses in one letter amino acid abbreviation): 8 (S), 35 (R), 48(V), 53(Q), 60(1), Date Recue/Date Received 2021-11-12 =
66(I), 67(N), 68((L), 89(L), 100(L), 108(E), 112(D), and 118 (L). Since human mature leptins elicit a biological response in monkeys, a 'pan, such as mature human leptin form 1 (SEQ ID
.NO:20) having one or more of the rhesus monkey divergent amino acids replaced with another amino acid, such as the amino acids in parentheses, may be employed in designing and preparing leptin analogs comprised by the chimeric polypeptides in accordance with the invention. It should be noted that certain rhesus divergent amino acids are also those found in, for example, the above mature marine leptin form I (positions 35, 68, 89, 100 and 112).
Thus, one may = prepare leptin analogs in which one or more amino acids at positions 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66,67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145 of, e.g., mature human leptin form 1 (SEQ ID NO:20) are replaced by the corresponding amino acid(s) at such position(s) in murine or rhesus monkey leptins (e.g.. SEQ
11) NO:2 and/or SEQ ID NO: 14).
101071 In accordance with the invention, chimeric polypeptide analogs may he designed and prepared to comprise contiguous regions of amino acids from human leptin analogs. In Some embodiments, the invention provides chimeric polypeptide analogs based on a wild type. seal leptin polypeptide wherein at least one contiguous region of 1-30 amino acids of a. wild type seal leptin sequence has been replaced with a contiguous region of 1-30 amino acids of a mature human leptin analog sequence, and wherein the mature human leptin analog sequence contains at least one amino acid substitution at a position where divergence is observed in a corresponding position in a leptin from another species. Chimeric poly-peptide analogs comprising two or more contiguous regions of 1-30 amino acids of a mature human leptin analog sequence are also provided.
1.0108j Chimeric polypeptides to which a chemical moiety is attached are polypeptide derivatives. Derivatization of chimeric polypeptides by attachment of one or more chemical moieties has been found to irovide some advantage under certain circumstances, such as increasing the stability and circulation time of the therapeutic protein and decreasing immunogenicity and propensity for, for example, generation of neutralizing antibodies and/or incidence of injection site reactions. See, e.g., WO 98/28427, US2007/0020284, U.S. Patent No.
4,179,337, Davis et al. , issued December 18, 1979. For a review, see Abuchowski eat , in =
Date Recue/Date Received 2021-11-12 ENZYMES AS DRUGS. (J. S. Holcerberg and J. Roberts, eds. pp. 367-383 (1981));
Francis et al., Id.
101.091 Polypeptide derivatives may constitute polypeptides to which a chemical modification has been made of one or more of its amino acid side groups, a-carbon atoms, terminal amino group, or terminal carboxylic acid 'group. A chemical modification includes, but is not limited to, attaching one or more chemical moieties, creating new bonds, and removing one or more chemical moieties. Modifications at amino acid side groups include, without limitation, alkylation, acylation, ester formation, amide formation, maleimide coupling, acylation of lysine &amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine.
Modifications of the terminal amino include, without limitation, the desamino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal amino include, without limitation, the desamino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications, such as alkyla.cyls, branched alkylacyls, alkylaryl-acyls. Modifications orthe terminal carboxy group include, without. limitation, the amide, lower alkyl amide, dialkyl amide, arylamide, alkylarylamidc and lower alkyl ester modifications. Lower alkyl is Ci-C4 alkyl. Furthermore, one or more side groups, or terminal groups, may be protected by protective groups known to the ordinarily-skilled synthetic chemist. The n.-carbon of an amino acid may be mono- or dimethylated.
[01101 Such derivatives include polypeptides conjugated to one or more water soluble polymer molecules, such as polyethylene glycol ("PEG") or fatty acid chains of various lengths (e.g., stearyl, palmitoyl, oclanoy1), by the addition of polyamino acids, such as poly-his, poly-arg, - poly-lys, and poly-ala, or by addition of small molecule substituents include short alkyls and constrained alkyls (e.g., branched, cyclic, fused, adamantyl), and aromatic groups. In some embodiments, the water soluble polymer molecules will have a molecular weight ranging from about 500 Daltons to about 60,000 Daltons.
101111 Such polymer-conjugations may occur singularly at the N- or C-terminus or at the side chains of amino acid residues within the sequence of a chimeric polypeptidc as disclosed herein.
Alternatively, there may be multiple sites of derivatization along the amino acid sequence of such a chimeric polypcptide. Substitution of one or more amino acids with lysinc, aspartic acid., gluiamic acid, or cysteine May provide additional sites for derivatization.
See, e.g., U.S. Patent =
Date Recue/Date Received 2021-11-12 Nos. 5,824,784 and 5,824,778. In sonic embodiments, a chimeric polypeptide may be conjugated to one, two, or three polymer molecules.
101.121 In some embodiments, the water soluble polymer molecules are linked to an amino, carboxyl, or thiol group, and may be linked by N or C termini, oral the side chains of lysine, aspartie acid, glutamic acid, or cysteine. Alternatively, the water soluble polymer molecules may be linked with diamine and dicarboxylic groups. In some embodiments, a chimeric polypeptide is conjugated to one, two, or three PEG molecules through an epsilon amino group on a lysine amino acid.
101131 Polypeptide derivatives also include polypeptides with chemical alterations to one or more amino acid residues. Such chemical alterations include amidation, glycosylation, acylation, sulfation, ph.osphorylation, acetylation, and cyclization. The chemical alterations may occur singularly at the N- or C-terminus or at the side chains of amino acid residues within the sequence of a leptin. In one embodiment, the C-terminus of these peptides may have a free -OH
or -NH7 group. In another embodiment, the N-terminal end may be capped with an isobutyloxycarhonyl group, an isopropyloxycarbonyl group, an n-butyloxycarbonyl group, an =
ethoxycarbonyl group, an isocaproyl group ("isocap"), an octanyl group, an octyl glycine group (denoted as "G(Oct)" or "octylGly"), art 8-aminooctanic acid group, a dansyl, and/or a Fmoc group. In some embodiments, cyclization can be through the formation of disulfide bridges.
Alternatively, there may be multiple sites of chemical alteration along the polypeptidc amino acid sequence.
(01141 fn certain embodiments, chimeric .polypeptides are chemically altered to include a Bolton-Hunter group. Bolton-Hunter reagents are known in the art ("Radioimmunoassay and related methods," A. E. Bolton and W. M. Hunter, Chapter 26 of HANDBOOK OF
EXPERIMENTAL
IMMUNOLOGY, VOLUME I, IMMtNOCHEMISTRY, edited by D. M. Wcir, Blackwell Scientific Publications, 1986), and may be used to introduce tyrosine-like moieties with a neutral linkage, through amino-terminal a-amino groups or 6-amino groups of lysine. In some embodiments, the N-tcnninal end of a polypeptide is modified with a Bolton-Hunter group. In some embodiments, an internal lysine residue is modified with a Bolton-Hunter group. In some embodiments, there may be multiple sites of Bolton-Hunter modification along the polypeptide amino acid sequence.
Bolton-Hunter reagents used for polypeptidc modification are commercially available, and may Date Recue/Date Received 2021-11-12 include, but are not limited to, water-soluble Bolton-Hunter reagent, Sullosuceinimidy1-3-14-hydrophenyllpropionate (Pierce Biotechnology, Inc., Rockford, IL) and Bolton-Hunter reagent-2, N-Succinimidyl 3-(4-hydroxy-3-iodophenyl) Priopionate (Wako Pure Chemical Industries, Ltd., Japan, catalog # 199-09341), An exemplary Bolton-Hunter group conjugated through an amide linkage to a polypeptide is illustrated below, wherein the dashed line passes through the amide bond:
=
HO

101151 Polypeptides may be iodinated (such as radiolabeled with 1251) before or after Bolton-Hunter modification,.
101161 Polypeptide derivatives may include one or more modifications of a "non-essential"
amino acid residue. In the context of the invention, a "non-essential" amino acid residue is a residue that can be altered, e.g., derivatized, without abolishing or substantially reducing the activity (e.g., the agonist activity) dike chimeric polypeptide. The chimeric polypeptides of the invention may include derivatizations of!, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues;
of these, one or more amino acid residues may be non-essential amino acid residues.
Additionally, the polypeptides of the invention may be derivatized such that they include additions of at least 1,2. 3, 4, 5,6, 7, 8, 9, 10 or more amino acids without abolishing or substantially reducing the activity of the polypeptide. Additionally, such non-essential amino acid residues may be substituted with an amino acid residue that is amenable to dcrivatization as described throughout.
101171 As used throughout, "amino Acid," "amino acid residue" and the like refer to natural amino acids, unnatural amino acids, and modified amino acids. Unless stated to the contrary, any reference to an amino acid, generally or specifically by name, includes reference to both the - ID and the L stercoisoiners if their structure allow such stercoisomeric forms. Natural amino acids include alanine (Ala), arginine (Arg), asparaginc (Asn), aspartie acid (Asp), eysteino(Cys).
glutamine (Gin), glutamic acid (Cilu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Lcu), Lysinc (Lys), inethionine (Met), phenylalanine (The), prolinc (Pro), scrinc (Ser), threonine Date Recue/Date Received 2021-11-12 =
=
(Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). Unnatural amino acids include, but are not limited to homolysine, hornoarginine, hornoserine, azetidinecarboxylic acid, 2-aminoudipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopirnelic acid, tertiary-hutylglycine, 2,4-diaminoisobutyric acid, desrnosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N- =
ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-.
hydroxyproline, isodesmoSine, allo-isoleucine, N-methylalanine, N-methylglycine,N-methylisoleucitte, N-methylpentylglycine, N-methylyaline, naphthalanine, norvaline., norleucine, omithine, pentylglycine, pipecolic acid and thioproline. Additional unnatural amino acids include modified amino acid residues which are chemically blocked, reversibly or irreversibly, or chemically modified on theirN-terminal amino group or their side chain groups, as for example, N-methylated D and L amino acids or residues wherein the side chain functional groups arc chemically. modified to another functional group. For example, modified =amino acids include rnethionine sulfoxide; methionine snlfone; aspartic acid- (beta-methyl ester), a modified amino acid of aspartic acid; N-eth.ylglyeine, a modified amino acid of glycine; or alanine carboxamide, a modified amino acid of alanine. Additional residues that can be incorporated are described in Sandberg et Med Chem. 41: 2481-91, 1998. =
= 101181 As mentioned above, chemical moieties suitable for such derivatization of the chimeric polypeptides include, for example, various water soluble polymers. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable. One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer/protein conjugate will be used therapeutically, and if so, the desired dosage, . circulation time, resistance to proteolysis, and other considerations.
For the chimeric polypeptides, the effectiveness of the derivatization may be ascertained by administering the derivatized polypeptide, in the desired form (Le., by osmotic pump, or, more preferably, by injection or infusion, or, further formulated for oral, pulmonary or nasal delivery, for example), and observing biological effects and biological responses as described herein.
=
=
101191 Such a water soluble polymer may be selected from the group consisting of, for example, polyethylene glycol, copolymers of ethylene glycol/propylene glycol, Date Recue/Date Received 2021-11-12 carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrolidone, poly-1, 3-dioxolanc, poly-1,3,6-trioxane, ethylene/rnaleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrolidone)polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohol. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. Also, suceinate, styrene, and hydroxyethyl starch may also be used.
10.1201 Derivatives of chimeric polypeptides in accordance with the invention may be prepared . = by attaching polyaminoacids or branch point amino acids. For example, the polyaminoacid may be an additional carrier protein, such as an Fe moiety, which can serve to also increase the circulation half life of the chimeric polypeptide. Additionally, such polyaminoacids may he selected from the group consisting of sentrn albumin (such as human serum albumin), an additional antibody or portion thereof (e.g. the Fe region), or other polyaminoacids, e.g.
polylvsines. As indicated below, the location of attachment of the polyaminoacid may be at the N-terminus of polypeptide, or C-terminus, or other places in between, and also may be connected by a chemical "linker" moiety to the polypeptide, such as a peptidic linker or a non.peptidic linker.
101211 The polymer may be of any molecular weight, and may he branched or unbranehect For polyethylene glycol, the preferred molecular weight is between about 2 kilodalions (kDa) and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. In certain embodiments, the polyethylene glycol is between about 2 kDa and = -about GO kDa. In certain embodiments, the polyethylene glycol is between about 2 kDa and about 40 kDa. In certain embodiments, the polyethylene glycol is between about 5 kDa and about 40 kDa. In certain embodiments, the polyethylene glycol is between about 10 kDa and about 40 kDa. In certain embodiments, the polyethylene glycol is between about 5 kDa and about 30 kDa. In certain embodiments, the polyethylene glycol is between about 5 kDa and about 20 kDa. In certain embodiments, the polyethylene glycol is between about 10 kDa and about 20=1cDa. Other sizes may he used, depending on the desired therapeutic profile (e.g., the = .
duration of sustained release desired, solubility characteristics, the effects, if any, on biological Date Recue/Date Received 2021-11-12 activity, the ease in handling, the degree or lack of antigenicity and other known effects ,of the polyethylene glycol attached to a leptin and/or to a chimeric polypeptide of the invention).
Additional considerations that may influence the selection of a-PEG of a particular molecular weight which may be attached to a chimeric.polypeptide to generate a chimeric derivative in accordance with the, invention include the extent to which such a molecular weight PEG may:
mitigate aggregation and/or increase the solubility of the chimeric poly-peptide, when present in a pharmaceutically acceptable composition or formulation, or when exposed to physiological fluids or tissues upon administration to a subject (such as by injection);
mitigate the incidence of injection site reactions caused by administration of the chimeric polypeptide upon administration to a subject by injection; mitigate the generation of neutralizing antibodies that may be raised against the chimeric polypeptide as a result of administration of such a chimeric polypeptide to a -subject; and the like.
[01221 The number of polymer molecules so attached may vary, and one skilled in the.art will =
be able to ascertain the resultant effect on function. One may mono-derivatize, or may provide for a di-, tri-, tetra- or some combination of derivatization, with the same or different chemical moieties (e.g., polymers, such as different weights of polyethylene glycols).
The proportion of polymer molecules to chimeric polypeptide molecules to be derivatized will vary, as will their .
concentrations in the reaction mixture. In general, the optimum ratio, in terms of efficiency of -reaction in that there is no excess unreacted chimeric polypeptide or polymer, will be determined by factors such as the desired degree of derivatization (e.g., mono, di-, tri-, etc.), the molecular . weight of the polymer selected, whether the polymer is branched or unbranched, and the reaction conditions.
101231 The chemical moieties should be attached to the chimeric polypeptide with consideration of the effects on functional or antigenic domains of the chimeric polypeptide.
There are a number of attachment methods available to those skilled in the art. E.g.; =
. EP 0 401 384 (coupling PEG to G-CSF), see also Malik et al., =
= .1992, Exit, Hematol. 20:1028-1035.(reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a.
reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free =
= 43 Date Recue/Date Received 2021-11-12 amino group may include lysine residues and the N-terminal amino acid residue.
Those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecule(s). Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
Attachment at residues important for receptor binding should be avoided if receptor, binding is desired.
101241 One may specifically desire to design and prepare N-terminally chemically modified chimeric polypeptides of the invention. Using polyethylene glycol as an illustration of the present compositions, one may select from a variety of polyethylene glycol.moleeules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to chimeric polypeptide molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N.-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective N-terminal chemical modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the .N-terininal) available for derivatization in a particular =
protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved. For example, one may selectively N-terminally pegylate the protein by performing the reaction at a pH which allows one to take advantage of the pKa differences between the c-amino group of the lysine residues and that of the u-antino group of the N.-terminal residue of the protein. By such selective derivatization, attachment of a water soluble polymer to a protein is controlled: the conjugation with the polymer takes place predominantly at the N-terminus of the protein and no significant modification of other reactive groups, such as the lysine side chain amino groups, occurs. Using reductive alkylation, the water soluble polymer may be of the type described above, and should have a single reactive aldehyde for coupling to the protein.
Polyethylene glycol propionaldehydc, containing a single reactive aldehyde, may be used.

Date Recue/Date Received 2021-11-12 la Methods of Design and Production [01251 Design of constructs. The chimeric polypeptides described herein can be designed at the amino acid level. These sequences can then be back translated using a variety of software products known in the an such that the nucleotide sequence is optimized for the desired expression host, e.g. based protein expression, codon optimization, restriction site content. For example, the nucleotide sequence can be optimized for E. twit based protein expression and for restriction site content. Based on the nucleotide sequence of interest, overlapping oligonueleotides can be provided for multistep PCR, as known in the art. These oligonucleotides can be used in multiple PCR reactions under conditions well known in the art to build the eDNA
encoding the protein of interest. For one example is IX AmplitaqTM Buffer, 1.3 mM MGC12, 200uM dNTPs, 4 U Amplitaq Gold; 0.2 uM of each primer (AmpliTaq Gold, ABI), with cycling pararneters: (94C:30s, 58C:1 min, 72C:lmin), 35 cycles.
101261 Restriction sites can be added to the ends of the PCR. products for use in vector ligation as known in the art. Specific sites can include Ndel and Xhol, such that the cDNA can then be in the proper reading frame in a pET45b expression vector (Novagen). By using these sites, any N-terminal His Tags that are in this vector can be removed as the translation start site would then be downstream of the tag. Once expression constructs arc completed, verification can be conduct by sequencing using e.g., T7 promoter primer, 1'7 terminator printer and standard ABI
BigDye Term v3.1 protocols as known in the art. Sequence information can be obtained. from e.g., an A13-1 3730 DNA Analyzer and can be analyzed using Vector NT! v.10 software (Invitrogen). Expression constructs can be designed in a modular manner such that linker sequences can be easily cut out and changed, as known in the art.
101271 Protease recognition sites, known in the art or described herein, can be incorporated into constructs useful for the design, construction, manipulation and production of recombinant chimeric poly-peptides described herein.
[01281 General methods of production. The chimeric polypeptides described herein may be prepared using biological, chemical, and/or recombinant DNA techniques that are known in the art. Exemplary methods are described herein and in US Patent No. 6,872,700-, WO
2007/139941; WO 2007/140284; WO 2008/082274; WO 2009/011544; and US
Publication No.
tts Date Recue/Date Received 2021-11-12 2007/0238669.
Other methods for preparing the compounds are set forth herein.
101291 The chimeric polypeptides described herein may be prepared using standard solid-phase peptide synthesis techniques, such as an automated or serMautotnated peptide synthesizer.
The chimeric polypeptides may be produced by non-biological peptide synthesis using amino acids and/or amino acid derivatives having reactive side-chains protected, the non-biological peptide synthesis comprising step-wise coupling of the amino acids and/or the amino acid derivatives to form a polypeptide according to the first aspect having reactive side-chains protected, removing the protecting groups from the reactive side-chains of the polypeptide, and folding of the polypeptide in aqueous solution. Thus, normal amino acids (e.g.
glycine, alanine, phenylalanine; isoleucine, leucine and valine) and pre-protected amino acid derivatives are used to sequentially build a poly-peptide sequence, in solution or on a solid support in an organic solvent. When a complete polypeptide sequence is built, the protecting groups are removed and the poly-peptide is allowed to fold in an aqueous solution.
101301 Typically, using such techniques, an alpha-N-carbatnoyl protected amino acid and an amino acid attached to the growing peptide chain on a resin are coupled at RT
in an inert solvent (e.g., dimethylformamide, N-mcthylpyrrolidinone, methylene chloride, and the like) in the presence of coupling agents (e.g., dicyclohexylcarbociiimide, 1-hydroxyhenzo-triazole, arid the like) in the presence of a base (e.g., diisopropylethylamine, and the like).
The alpha-N-carbamoyl protecting group is removed from the resulting peptide-resin using a reagent (e.g., trifluoroacetic acid, piperidine, and the like) and the coupling reaction repeated with the next =
desired N-protected amino acid to be added to the peptide chain. Suitable N-protecting groups are well known in the art, such as t-butyloxyearbonyl (am) fluorenylmethoxycarbonyl (Fmoc), and the like. The solvents, amino acid derivatives and 4-methylbenzitydryl-arnine resin used in the peptide synthesizer may be purchased from Applied Biosystems Inc. (Foster City, Calif.).
101311 For chemical synthesis solid phase peptide synthesis can be used for the chimeric polypeptides, since in general solid phase synthesis is a straightforward approach with excellent scalability to commercial scale. Solid phase peptide synthesis may be carried out .with an automatic peptide synthesizer (Model 430A, Applied Biosysterns inc., Foster City, Calif) using the NMP/H013t (Option I) system and Woe or Frnoc chemistry (.S'ee Applied Biosystems User's Date Recue/Date Received 2021-11-12 Manual for the ABT 430A Peptide Synthesizer, Version 1.38 Jul. 1, 1988, section 6, pp. 49-70, Applied Biosystems, Inc., Foster City, Calif.) with capping. 13oc-peptide-resins may be cleaved with TIF (-5 C to 0 C, I hour). The peptide may be extracted from the resin with alternating water and acetic acid, and the filtrates lyophilized. The Fmoc-peptide resins may be cleaved .
according to standard methods (e.g., Introduction to Cleavage Techniques, Applied Biosystems, Inc., 1990, pp. 6-12). Peptides may also be assembled using an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).
101321 The compounds described herein may also he prepared using recombinant DNA
techniques using methods known in the art, such as Sambruok et al., 1989, MOLECULAR
CLONING: A LA 130KATORY MANnint., 2d Ed., Cold Spring Harbor. Non-peptide compounds may be prepared by an-known methods. For example, phosphate-containing amino acids and peptides containing such amino acids, may be prepared using methods known in the. art, such as described in Bartlett et al, 1986, Biorg. Chain. 14:356-377.
[01331 The chimeric polypeptides may alternatively be produced by recombinant techniques well known in the an See, e.g., Sambrook et al., 1989. (Id.). These chimeric polypeptides produced by recombinant technologies may be expressed from a polynucleutide.
One skilled in the art will appreciate that the polynueleotides, including DNA and RNA, that encode such chimeric polypeptides may be obtained from the wild-type cDNA, e.g. human leptin, taking into consideration the degeneracy of codon usage, and may further be engineered as desired to incorporate the indicated substitutions. These polynucleotide sequences may incorporate codons facilitating transcription and translation of mRNA in microbial hosts. Such manufacturing sequences may readily be constructed according to the methods well known in the art. See, e.g., WO 83/04053, The polynuelentides above may also optionally encode an N-terminal methionyl residue. Non-peptide compounds useful in the present invention may be prepared by art-known methods. For example, phosphate-containing amino acids and peptides containing such amino acids may be prepared using methods known in the art. See, e.g., Bartlett and Landen, 1986, Bior g..Chem.
14: 356-77.
101341 A variety of expression vector/host systems may be utilized to contain and express a chimeric polypeptide coding sequence. 'These include, but are not limited to, microorganisms Date Recue/Date Received 2021-11-12 =
=
such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA .
expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaIvIV; tobacco mosaic virus, TIv11/) or =
transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems. Mammalian cells that are useful in recombinant protein productions include, but are = not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such =
as COS-7), WI 38, BHK, HepG2, 313,=REN, MOCK, A549, PC 12, K562 and 293 cells.
Exemplary protocols for the recombinant expression of the protein are described herein and/or = are known in the art.
101351 As such, polynueleotide sequences are useful in generating new and useful viral and plasmid DNA. vectors, new and useful transformed and transfeeted prokaryotic and eukaryotic host cells (including bacterial, yeast, and mammalian cells grown in culture), and new and useful methods for cultured gowth of such host 'cells capable of expression of the present chimeric polypeptides.- The polynucleotide sequences encoding chimeric polypeptides herein may be useful for gene therapy in instances where underproduction of chimeric polypeptides would be alleviated;or the need for increased levels of such would be met.
=
[01361 The present invention also provides for processes for recombinant DNA
production of the present chimeric poly-peptides. Provided is a process for producing the chimeric polypeptides from a host cell containing nucleic acids encoding the chimeric polypeptide comprising: (a) culturing the host cell containing polynucleotides encoding the chimeric polypeptide under conditions facilitating the expression of the DNA molecule; and (b) obtaining the chimeric polypeptide.
[01371 Host cells may be prokaryotic or eukaryotic and include bacteria, mammalian cells (such as Chinese Hamster Ovary (CHO) cells, monkey cells, baby hamster kidney cells, cancer = cells or other cells), yeast cells, and insect cells.
[01381 Mammalian host systems for the expression of the recombinant protein also arc well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited Date Recue/Date Received 2021-11-12 =
to, acetylation, earhoxylation, glycosylation, phasphorylation, lipidation and acylation. Post-translational processing, which cleaves a "prepro" form of the protein, may also he important for correct insertion, folding and/or function. Different host cells, such as CHO, lieta, MDCK, 293, WI38, and the like, have specific cellular machinery and characteristic mechanisms for such post-translational activities, and may be chosen to ensure the correct modification and processing of the introduced foreign protein.
(01391. Alternatively, a yeast system may be employed to generate the chimeric polypeptities of the present invention. The coding region of the chimeric polypeptides DNA is amplified by ' PCR. A DNA encoding the yeast pre-pro-alpha leader 6equence is amplified from yeast genomie DNA in a PCR reaction using one primer containing nucleotides 1-20 of the alpha mating factor gene and another primer complemental)/ to nucleotides 255-235 of this gene (Kuijan and.
Herskowitz, 1982, Cell, 30:933-43). The pre-pro-alpha leader coding sequence and chimeric polypeptide coding sequence fragments are ligateci into a plasmid containing the yeast alcohol dehydrogenase (ADH2) promoter, such that the promoter directs expression of a fusion protein consisting of the pre-pro-alpha factor fused to the mature chimeric polypeptide. As taught by Rose and Broach, Meth. E172. 185: 234-79, Goeddel ed., Academic Press, Inc., San Diego, California (1990), the. vector further includes an ADH2 transcription terminator downstream of the cloning site, the yeast "2-micron" replication origin, the yeist leu-2d gene, the yetst REP1 and REP2 genes, the E. coli beta-lacianase gene, and an E. cob origin of replication. The beta-laetamase andleu-2d genes provide for selection in bacteria and yeast, respectively. The leu-2d gene also facilitates increased copy number of the plasmid in yeast to induce higher levels of expression. The REP' and RE.P2 genes encode proteins involved in regulation of the plasmict copy number.
10140.1 The DNA construct described in the preceding paragraph is transformed into yeast cells using a known method, e.g., lithium acetate treatment (Steams et at., I990,.
Meth. Enz. 185: 280-297), The ADH2 promoter is induced upon exhaustion of glucose in the growth media (Price et a)., 1987. Gene 55:287). The pre-pro-alpha sequence effects secretion of the fusion protein from.
the cells. Concomitantly, the yeast KE.X2 protein cleaves the pre-pro sequence from the mature chimeric polypeptides (Bitter et at., 1984, Proc. Nod. /lead Sci. USA 81:5330-5334).

Date Recue/Date Received 2021-11-12 101411 Chimeric polypcptides of the invention may also be recombinantly expressed in yeast, e.g., Pichia. using a commercially available expression system, e.g., the Pichia Expression System (Invitrogen, San Diego, California), following the manufacturer's instructions. This system also relics on the pre-pro-alpha sequence to direct secretion, but transcription of' the insert is driven by the alcohol oxidase (AOX1) promoter upon induction by methanol.
The secreted chimeric polypeptide is purified from the yeast growth medium by, e.g., the methods used to purify said chimeric polypeptide from bacterial and mammalian cell supernatants.
101421 Alternatively, the DNA encoding a chimeric polypeptide may he cloned into a baculovirus expression vector, e.g. pVL1393 (PharMingean Diego, California).
This chimeric-polypeptide-encoding vector is then used according to the manufacturer's directions (PharMingen) or known techniques to infect Spodopierafriigiperda cells, grown fir example in =
sF9 protein-free media, and to produce recombinant protein. The protein is purified and = concentrated from the media using methods known in the art, e.g. a heparin-Sepharose column = (Pharmacia, Piscataway, New Jersey) and sequential molecular sizing columns (Amieon, Beverly, Massachusetts), and resuspended in appropriate solution, e.g. PBS.
SDS-PAGE =
analysis can be used to characterize the protein, for example by showing a single band that confirms the size of the desired chimeric polypeptide, as can full amino acid amino acid = sequence analysis, e.g. Edman sequencing on a Proton 2090,Pcptide Sequencer, or confirmation of its N-terminal sequence.
101431 For example, the DNA sequence encoding the predicted mature chimeric polypeptide may be cloned into a plasrnid containing a desired promoter and, optionally, a leader sequence (see, e.g., Better et at., .1988. Science 240:1041-1043). The sequence of this construct may be =
confirmed by automated sequencing. The plasmid is then transformed into E.
coil, strain MC1061, using standard procedures employing CaCl2 incubation and heat shock treatment of the bacteria (Sainbrook at at., Id). The transformed bacteria are grown in LB
medium supplemented with carbenicillin, and production of the expressed protein is induced by growth in a suitable medium'. If present, the leader sequence will affect secretion of the mature chimeric polypeptide and be cleaved during secretion. The secreted recombinant chimeric polypeptide is purified from the bacterial culture media by the method described herein.
Date Recue/Date Received 2021-11-12 (01441 Alternatively, the chimeric polypeptides may be expressed in an insect system. Insect systems for protein expression are well known to those of skill in the art. En one such system, Autographa californica nuclear polyhedrosis virus (AcIslPy) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in .Trichoplacia larvae. The chimeric polypeptide coding sequence is cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
Successful insertion of a chimeric polypeptide will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S.
frugiperda cells or Trichoplusia larvae in which a chimeric polypeptide of the present invention is expressed (Smith et at., 1983,J. Virol. 46:584; Engelhard et at., 1994, Proc. Natl. Acad. Sei.
USA 91:3224-3227).
101451 In another example, the DNA sequence encoding the chimeric polypeptides-may be amplified by PCR and cloned into an appropriate vector, for example, pGEX-3X.(P.harrnacia, Piscataway, New Jersey). The pGEX vector is designed to produce a fusion protein comprising glutathione-S-transferase (CST), encoded by the vector, and a protein encoded by a DNA
fragment inserted into the vector's cloning site. The primers for the PCR may be generated to include, for example, an appropriate cleavage site. The recombinant fusion protein may then be cleaved from the GST portion of the fusion protein. The pGEX-3X/ chimeric polypeptide construct is transformed into E. coil XL-1 Blue cells (Stratagene, La Jolla, California), and individual transforrnants are isolated and grown at 37"C in LB medium (supplemented with carbenicil lin) to an optical density at wavelength 600 nm of 0.4, followed by further incubation for 4 hours in the presence of 0.5 rriM Isopropyl beta-D-th.iogalactopyranoside (Sigma Chemical Co., St. Louis, Missouri). Plasmid DNA from individual trans fonnants is purified and partially sequenced using an automated sequencer to confirm the presence of the desired chimeric polypeptide-encoding gene insert in the proper orientation.
=
(0146j The fusion protein, when expected to he produced as an insoluble inclusion body in the bacteria, may be purified as described above or as follows. Cells are harvested by centrifugation;
washed in 0.15 M NaCI, 10 infv1 iris, pH 8, I rnIvl EDTA; and treated with 0.1 mentL lysozyme (Sigma Chemical Co.) for 15 mm. at RT. The lysate is cleared by sonication, and cell debris is pelleted by centrifugation for 10 min. at 12,000xg. The fusion protein-containing pellet is resuspended in 50 m114 iris, pH 8, and 10 nuM EDTA, layered over 50% glycerol, and = 51 = =
Date Reeue/Date Received 2021-11-12 centrifuged for 30 min. at 6000xg. The pellet is resuspended in standard phosphate buffered saline solution (PBS) free of Mg++ and Ca++. The fusion protein is further purified by fractionating the resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook et al., supra). The gel is soaked in 0.4 M KC1 to visualize the protein, which is excised and electroeluted in gel-running buffer lacking SDS. If the GST/chimeric polypeptide fusion protein is produced in bacteria as a soluble protein, it may be purified using the GST
Purification Module (Pharmacia Biotech).
[01471 The fusion protein may be subjected to digestion to cleave the (.ST
from the mature chimeric poly-peptide. The digestion reaction (20-40 ttg fusion protein, 20-30 units human ihrontbin (4000 LI/ing (Sigma) in 0.5 mL PBS) is incubated 16-48 hrs. at RT
and loaded on a denaturing SDS-PAGE gel to fractionate the reaction products. The gel is soaked in 0.4 M KCI
. to visualize the protein bands. The identity of the protein band corresponding to the expected molecular weight of the chimeric polypeptide may be confirmed by partial amino acid sequence = analysis using an automated sequencer (Applied 13iosystems Model 473A.
Foster City, Cali font ia).
101481 In a particularly exemplary method of recombinant expression of the chimeric polypeptides of the present invention, 293 cells may be co-transfected with plasmids containing the chimeric polypeptides cDNA in the pCMV vector (5' CMV promoter, 3' MIT
poly A
sequence) and pSV2neo (containing the neo resistance gene) by the calcium phosphate method.
In one embodiment, the vectors should be linearized with Scat prior to tran.sfection. Similarly, an alternative construct using a similar pC1v1V vector with the neo gene incorporated can be -, =
used. Stable cell lines are selected from single cell clones by limiting dilution in growth media containing 0.5 mg/mL G418 (neomycin-like antibiotic) for 10-14 days. Cell lines are screened for chimeric polypeptides expression by ELBA or Western blot, and high-expressing cell lines are expanded for large scale growth.
101491 It is preferable that the transformed cells are used for long-term, high-yield protein production and as such stable expression is desirable. Once such cells are transformed with vectors that contain selectable markers along with the desired expression cassette, the cells may , be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The selectable marker is designed to confer resistance to selection, and its presence Date Recue/Date Received 2021-11-12 allows growth and recovery of cells that successfully express the introduced sequences. =
Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell.
101501 A number of selection systems may be used to recover the cells that have been=
transformed for recombinant protein production. Such selection systems include, but are not limited to, liSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltrimsferase genes, in tic-, hgprt- or aprt- Fclls, respectively.
Also, anti-metabolite resistance can be used as the basis of selection for dhfr, that confers resistance to methotrexate;
= - gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside, G418; also, that confers resistance to chlorsulfuron; and hygro, that confers . resistance to hygromycin. Additional selectable genes that may be useful include trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine. Markers that give a Visual indication for identification of transformants include anthocyanins, beta-glucuronidase and its substrate, GUS, and lucilerase and its substrate, Nei fen n.
101511 The chimeric polypeptides of the present invention may be produced using a combination of both automated peptide synthesis and recombinant techniques.
For example. a chimeric polypeptide of the present invention may contain a combination of modifications including deletion, substitution, insertion and derivatization by PEGylation (or other moiety, e.g.
polymer, fatty acyl chain, C-terminal amidation). Such a chimeric polypeptide may be produced in stages. In the first stage, an intermediate chimeric polypeptide containing the modifications of deletion, substitution, insertion, and any combination thereof, may be produced by recombinant techniques as described. Then after an optional purification step as described herein, the intermediate chimeric polypeptide is PEGylated (or subjected to other chemical derivatization,-, = e.g., acylation, C-terminal amidation) through chemical modification with an appropriate =
PEGylating reagent (e.g. from NeKtarTM Transforming Therapeutics, San Carlos, California) to yield the desired chimeric polypeptide derivative. One skilled in the art will appreciate that the above-described procedure may be generalized to apply to a chimeric polypeptide containing a combination of modifications selected from deletion, substitution, insertion, derivation, and other means of modification well known in the art and contemplated by the present invention.

=
Date Recue/Date Received 2021-11-12 =
101521 Peptides may be purified by any number of methods known in thc art, including as described herein In one method peptides are purified by RP-IIPLC (preparative and analytical) using a Waters Delta Prep 3000 system. A C4, C8 or C18 preparative column (I
Ofl, 2.2X25 cm;
VydacTm, Hesperia, -Calif) May be used to isolate peptides, and purity may be determined using a = C4, C8 or CIS analytical column 5u 0.46X25 cm; VydacTm). Solvents (A=0.1%
TFA/water and B=0.1 6 TFA/CH5CN) may be delivered to the analytical column at a flow rate of 1.0 ml/min and to the preparative column at 15 mlintin. Amino acid analyses may be performed on the Waters Pico Tag system and processed using the Maxima program. Peptides may be hydrolyzed by vapor-phase acid hydrolysis (115C, 20-24 h). Flydrolysates may be derivatized and analyzed by standard methods (Cohen et al, THE Pico TAG METHOD: A MANUAL OF ADVANCED
TECHNIQUES FOR AmiN0 ACto ANALYStS, pp. 11-52, MilliporeTM,Corporation, Milford, Mass.
(1989)). Fast atom bombardment analysis may be carried out by M-Scan, Incorporated (West Chester, Pa.). Mass calibration may be performed using cesium iodide or cesium iodide/glycerol. Plasma desorption ionization analysis using time of flight detection may be carried out on an Applied Biosystems Bio-Ion 20 mass spectrometer.
101531 Chimeric polypeptide expression assay. Methods are available for assaying the level of protein expression by a host cell. Procedures useful for assaying the level of protein expression by a host cell are exemplified in the following typical protocol.
About 25 ttIBL21 E. coil cells are transformed with 2u1plastnid DNA (expression vector for the chimeric polynucleotide). Cells can be plated and incubated overnight at 37 degrees C
or at room temperature (RI) over a 48-hr period. A single colony can be selected and used to grow starter culture in 4 ml LB media with appropriate antibiotic for hrs. Glycerol stocks can be prepared by adding 100u180% sterile glycerol to 900111 stock, which can then be mixed gently and stored at -SOC. A 250 it sample can be removed for TCP uninduced sample. An aliquot, for example, 2 ml of Magic media containing appropriate antibiotic can be inoculated with 5 n1 starter culture, which can then be incubated overnight (up to 24 hrs) at 37C, 300 rpm. As known in the art, Magic Media is autoindueing. Alternatively, 60 ml Magic Media containing appropriate antibiotic can be inoculated with 60 I starter culture in a 250m1 or 125 ml Thompson flask, which can then be incubated overnight (up to 24 his) at 30C, 300rprn. After incubation, 250 ol culture can be removed from each tube and the cells petleted. The cell can be resuspended in ml 50 mM Tris pH 8, 150rruM NaCI, to which can be added 0.1 volumes (100u1) POP culture Date Recue/Date Received 2021-11-12 reagent and 1 n1 r-lysozyme (1:750 dilution in r-lysozyme buffer). The mixture can be mixed well and incubated at least 10 min at RT. The preparation can then be centrifuged 10 min at 14000 x G. The supernatant (soluble fraction) can be removed and retained, and samples can be prepared for gel analysis (15 III + 5 al LDS). The remaining inclusion body pellet can be resuspended in 1ml 1% SDS with sonication. The sample can be prepared for gel analysis (15u1+ 5 nt LDS). For uninduced samples, 1.0 volumes POP culture reagent and I
nl r-lysozyme (1:750 dilution in r-lysozyme buffer) can be added. The mixture can be mixed well arid incubated at least 10 min at RI. These samples may not need Lobe centrifuged. The sample can then 'be prepared for gel analysis (15111 5 RI LDS). NU-PAGE gels (4-12%) non-reduced in 1X MES buffer can be run and stained with SirnplyBlueTM microwave protocol.
Destaining can be conducted overnight, as known in the art. A gel image can be retained, and analyzed to determine protein expression levels.
101541 Inclusion Body preparation. For chimeric polypcptides that are found in the inclusion body fraction, the following procedure can be beneficial. The cell pellet can be resuspended in a minimum of 100 ml Lysis buffer for each 50 ml culture. Upon the. addition of 30m1, a I Ornl pipette can be used to resuspend. then the tube can be washed out with an additional 70m1. The resuspended cell solution can be multiply run, e.g., 4'passes, through a microfluidizer at 100 PSI
(min) taking care to keep chamber in ice water through the entire process. The fluidized slurry can be centrifuged at 14000 x g, 20 min (e.g., JI.A 10.5, 10,000mm, using 250 ml Nalgene bottles). The inclusion body pellet can he resuspended on ice in chilled lysis hurler with stir bar and stir plate for 1 hour at 4C after disruption with pipette tip. The pellet earl be resuspended a second time in distilled WO with stir bar and stir plate for 1 hour at 4C
after disruption with pipette tip, followed by centrifugation at 14000 x g, 15 min. The supernatant can be removed and discarded. The resultant can be stored at -80C.
101551 Protein purification. As described herein, numerous methods are known for isolation of expressed polypeptides. The following is one example. Inclusion body pellets can be solubilized in appropriate volume of sohibilization buffer (8M urea or 8M
guanidine, 50 tnM
iris, 10 mM DTT, pH 7.75) ibr 1 hour at RT. The solubilized pellets can be centrifuged for 20 min at 27 000g. Filtered (e.g., 0.4 -um) supernatant can be transferred drop by drop into appropriate volume of refolding buffer (50 inM Tris-HCI, 1 M urea, 0.8 M
arginine, 4 mM
. 55 =
Date Recue/Date Received 2021-11-12 cysteine, 1 mM cystarnine; pH 8) at RT. The result can then be placed at 4 C
overnight or longer with gentle mixing. Samples can be concentrated and run on a gel filtration column (SuperdexTm 75 26160) at 1-2 ml/min in 4C environment using a GE
Healthsciences AKTAFPLGm. Appropriate protein containing fractions can be identified via SDS-PAGE, pooled and run through a second gel filtration column. Pooled protein can then be concentrated in Arnicon filter to appropriate concentration and assayed for endotoxin levels using, e.g., Endosafet PI'S Reader (Charles River), as known in the an. Once a protein sample has passed the endotoxin criteria, it can be sterile filtered, dispensed into aliquots and run through quality control assays. Quality control assays can include analytical HPLC-SEC, non reducing SDS
PAGE and RP HPLC ¨ MS to obtain approximate mass. Proteins can be obtained in lx PBS (137 rnlvi sodium chloride, 2.7 mM potassium chloride, 4.3 triM disodiurn phosphate, 1.4 mM
tnonopotassium phosphate, pH7.2), distributed into aliquots and flash frozen for storage at -70 to -80 'C.
IV. Methods of Use and Treating Disease =
101561 Indications. A variety of diseases and disorders are contemplated to be beneficially treated by the polypeptide compounds and methods described herein.
101571 Obesity and overweight. Obesity and its associated disorders including overweight are common and serious public health problems in the United States and throughout the world.
Upper body obesity is the strongest risk factor known for type 2 diabetes mellitus arid is a strong risk factor for cardiovascular disease. Obesity iS a recognized risk factor for hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystie ovarian syndrome, cancers of the breast, prostate, and colon, and increased incidence of complications of general anesthesia. See, e.g., Kopelman, 2000õ Nature 404:635-43.
101581 Obesity reduces life-span and carries a serious risk of the co-morbidities listed above, as well disorders such as infections, varicose veins, acanthosis nigricans, eczema, exercise intolerance, insulin resistance, hypertension hypercholesterolemia, cholelithiasis, orthopedic injury, and thromboembolic disease. See e.g,, Rissanen et al, 1990, Br. Med.
J., 301:835-7.
Obesity is also a risk factor for the group of conditions called insulin resistance syndrome, or Date Recue/Date Received 2021-11-12 "Syndrome X" and metabolic syndrome. The worldwide medical cost of obesity and associated disorders is enormous.
[01591 The pathogenesis of obesity is believed to be multi-factoral. A.
problem is that, in obese subjects, nutrient availability and energy expenditure do not come into balance until there is excess adipose tissue. The central nervous system (CNS) controls energy balance and coordinates a variety of behavioral, autonomic and endocrine activities appropriate to the metabolic status of the animal. The mechanisms or systems that control these activities are broadly distributed across the forebrain (e.g., hypothalamus), hindbrain (e.g., brainstem), and spinal cord. Ultimately, metabblie (i.e., fuel availability) and cognitive (i.e., learned preferences) information from these systems is integrated and the decision to engage in appetitive (food seeking) and consummatory (ingestion) behaviors is either turned on (meal procurement and initiation) or turned off (meal termination). The hypothalamus is thought to be principally responsible for integrating these signals and then issuing commands to the brainstem. Brainstein nuclei that control the elemems of the consummatory motor control system (e.g., muscles =
responsible forchewing and Swallowing). As such, these CNS nuclei have literally been referred to as constituting the "final common pathway" for ingestive behavior.
101601 Neuroanatomical and pharmacological evidence support that signals of energy and = nutritional homeostasis integrate in forebrain nuclei and that the consummatoty motor control system resides in brainstem nuclei, probably in regions surrounding the trigeminal motor = nucleus. There are extensive reciprocal connection between.the hypothalamus and brainstem.
A variety of CNS-directed anti-obesity therapeutics (e.g., small molecules and peptides) focus predominantly upon forebrain substrates residing in the hypothalamus and/or upon hindbrain . substrates residing in the brainstem. =
[01611 Obesity remains a poorly treatable, chronic, essentially intractable metabolic disorder.
Accordingly, a need exists for new therapies useful in weight reduction and/or weight maintenance in a subject. Such therapies would lead to a profound beneficial effect on the subject's health. Methods and therapies employing the chimeric peptides disclosed herein, either alone or in combination with other anti-obesity agents (see, e.g., WO
2009064298 and US 20080207512) may.provide such beneficial effects.
=

=
Date Recue/Date Received 2021-11-12 = 2 10162j Leptin deficiency. Leptin deficiency has been shown to result in obesity. One form of leptin deficiency is congenital leptin deficiency, a rare genetic disorder.
See Montague et al., .1997, Nature 387: 903-908. Severe leptin deficiency can be a result of uncontrolled insulin-deficient diabetes mellitus that results from destruction of insulin-secreting 13-ce1ls. It is theorized that the lack of insulin leads to synthesis and storage of triglycerides in adipose tissue, which prevents weight gain and in turn dramatically reduces plasma leptin levels since leptin is synthesized in adipose tissue. These and other Leptin deficiencies, and disease and disorders that result from such deficiencies, can be treated with leptin replacement therapy, such as via daily leptin or leptin agonist injections. The chimeric polypeptides described herein can provide a .
- more convenient and advantageous therapeutic treatment of such diseases and disorders.
(01631 Diabetes and cardiovascular disease. Diabetes mellitus is recognized as a complex, chronic disease in which 60% to 70% of all case fatalities among diabetic patients are a result of cardiovascular complications. Diabetes is not only considered a coronary heart disease risk equivalent but is also identified as an independent predictor of adverse events, including =
recurrent myocardial infarction, congestive heart failure, and death following a cardiovascular incident. The adoption of tighter glucose control and aggressive treatment for cardiovascular risk factors would be expected to reduce the risk of coronary heart disease complications and improve overall survival among diabetic patients, Yet, diabetic patients are two to three times more likely to experience an acute myocardial infarction than non-diabetic patients, and diabetic patients live eight to thirteen years less than non-diabetic patients.
f0164J Understanding the high risk nature of diabetic/acute myocardial infarction patients, the American College of Cardiology/American Heart Association ("A.CC/AHA") clinical practice = guidelines for the management of hospitalized patients with unstable angina or non-ST-elevation myocardial infarction (collectively referred to as "ACS") recently recognized that hospitalized diabetic patients are a special population requiring aggressive management of hyperglycemia.
Specifically, the guidelines state that glucose-lowering therapy for hospitalized diabeticIACS
. patients should be targeted to achieve preprandial glucose less than 10 mg/dL, a maximum daily target than 189 mg/dL, and a post-discharge hemoglobin Ale less than 7%.
101651 In a nationwide sample of elderly ACS patients, it was demonstrated that an increase in =
30-day mortality in diabetic patients corresponded with the patients having higher glucose values =

Date Recue/Date Received 2021-11-12 upon admission to the hospital. See "Diabetic Comnary Artery Disease &
Intervention,"
Coronary Therapeutics 2002, Oak Brook, IL, September 10, 2002. There is increasing evidence that sustained hyperglycemia rather than transient elevated glucose upon hospital admission is related to serious adverse events. Although the ideal metric for hyperglycemia and vascular risk in patients is not readily known, it appears that the mean glucose value during hospitalization is most predictive of mortality. In a separate study of ACS patients form over forty hospitals in the United States, it was found that persistent hyperglycemia, as opposed to random glucose values upon admission to the hospital, was more predictive of in-hospital mortality.
See Acute Coronary Syndrome S'unimit: ,4 State of the An Approach, Kansas City, MO, September 21, =
2002. Compared with glucose values upon admission, a logistic regression model of glucose control over the entire hospitalization was most predictive of mortality.
There was nearly a two-fold increased risk of mortality during hospitalization for each 10 mgidL
increase in glucose over = 120.meille In a smaller cohort of consecutive diabeticiACS patients, there was a graded increase in mortality at one year with. increasing glucose levels upon hospital admission. In the hospital setting, the ACC/AH.k guidelines suggest initiation of aggressive insulin therapy to achieve lower blood glucose during hospitalization.
101661 It has been reported that leptin can have direct benefit to treating diabetes, particularly in type I diabetes and type 11 diabetes, with or without the presence of obesity, and more particularly in conditions ()flow serum leptin. It has been reported that leptin replenishment reduced or prevented hyperinsulinemia, insulin resistance and hyperglycemia in various animal models of' diabetes type 1 and 2 with or without attendant obesity. For example, high lepti.a =
plasma levels generated either by pharmacological adnainistration of leptin or with adenoviral gene therapy reduced hyperglycemia and associated increases of plasma glueagon levels in STZ-induced diabetes, despite persistently low insulin levels.
(01471 Lipid regulation diseases. As known in the art lipodystrophy is characterized by abnormal or degenerative conditions of the body's adipose tissue. Dyslipidemia is a disruption in the normal lipid component in the blood. It is believed that prolonged elevation of insulin levels can lead to dyslipidernia. Hyperlipidemia is the presence of raised or abnormal levels of lipids andlor lipoproteins in the blood. Hypothalamic amenorrhea is a condition in which menstruation = stops for several months due to a problem involving the hypothalamus. It has been found that Date Recue/Date Received 2021-11-12 =
=
leptin replacement therapy in women with hypothalamic amcnorrhea improves reproductive, thyroid, and growth hormone axes and markers of bone formation without causing adverse effects. See e.g., Oral et al., N Engl j Med. 2004, 351: 959-962, 987-997.
Fatty liver disease, e.g., nonalcoholic fatty liver disease (NAFLD) refers to a wide spectrum of liver disease ranging from simple fatty liver (steatosis), to nonalcoholic steatohepatitis (NASH), to cirrhosis (irreversible, advanced scarring of the liver). All of the stages of NAFLD
have in common the accumulation of fat (fatty infiltration) in the liver eellS(hepatocytes). It is believed that leptin is one of the key regulators for inflammation and progression of fibrosis in various chronic liver diseases including NASH.. See e.g., Ikejima et al., llepatology Res. 33:151-154.
=
101681 Additionally, without wishing to be hound by any theory, it is believed that relative insulin deficiency in type 2 diabetes, glucose toxicity, and increased hepatic free fatty acid burden through elevated delivery from intra-abdominal adipose tissue via the portal vein, are implicated as possible causes in fatty liver disorders. Indeed, it has been hypothesized that eating behavior is the key factor driving the metabolic syndrome of obesity with its many corollaries, including NASH.. Accordingly, treatments aimed at decreasing food intake and increasing the number of small meals, as has already been demonstrated in type 2 diabetes, may effectively treat and prevent NASH. Drugs that promote insulin secretion and weight loss, and delay gastric emptying are also effective at improving glucose tolerance and thus may improve fatty liver with its attendant hyperinsulinemia. Thus, use of a chimeric leptin polypeptide can be well suited as a treatment modality for this condition. Accordingly, chimeric polypeptides described herein can be useful in the treatment of fatty liver disorders.
101691 Alzheimer's disease. Alzheimer's disease (AD), as known in the art, is associated with plaques and tangles in the brain which include dysregulation.of the A-beta protein. It is believed that brain lipids are intricately involved in A-beta-related pathogenic pathways, and that =
an important modulator orlipid homeostasis is leptin. Accordingly, leptin can modulate bidirectional A-beta kincsis, reducing its levels extracellularlv. Indeed, it has been demonstrated = that chronic administration of leptin to A D-transgenic animals reduced the brain A-beta load, underlying its therapeutic potential. See Fewlass et al., 2004, FASEB J., 18:1870-1878.
Additionally, type 2 diabetes mellitus and AD share epidemiological and biochemical features in that both are characterized by insoluble protein aggregates with a fibrillar conformation ¨ amylirt Date Recue/Date Received 2021-11-12 =
in type 2 DM pancreatic islets, and Aft in AD brain. Without wishing to be bound by any theory, it is believed that similar toxic mechanisms may characterize type-2 DM and AD, See Lim eta)., FEBS Lett, 582:2188-2194.
[01701 Metabolic syndrome X. Metabolic Syndrome X is characterized by insulin resistance, dyslipidemia, hypertension, and visceral distribution of adipose tissue, and plays a pivotal role in the pathophysiology of type 2 diabetes. It has also been found to he strongly correlated with NASH, fibrosis, and cirrhosis of the liver. Accordingly, chimeric polypcptides described herein can be useful in the treatment of metabolic syndrome X.
[01711 Huntington's Disease. Huntington's Disease is an aittosornal dominant, =regenerative disease. Features of the disease include motor disturbances, dementia, psychiatric problems, and unintended weight loss. Chimeric polypeptides described herein can be useful in the treatment of Huntington's Disease.
[01721 Accordingly, in one aspect, there is provided a method for treating a disease or disorder in a subject. The subject is in need of treatment for the disease or disorder.
The disease or disorder can be lipodystrophy, dyslipidemia, hyperlipideinia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease or diabetes (including type 1 and type 10. Additional diseases and disorders which can be treated by the compounds and methods described herein include nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X and Huntington's Disease. The method of treatment includes administration to the subject of a chimeric polypeptide as described herein in an amount effective to treatment the disease or disorder.
V. Assays 101731 Methods for production and assay of chimeric polypeptides described herein are generally available to the skilled artisan. Further, specific methods are described herein as well as in the patent publications and other references cited herein,' 101741 Food intake. Without wishing to be bound by any theory, it is believed that food intake is useful in the assessment of the utility of a compound as described herein. For example, it is known that a number of metabolic pathologies are related to food intake (e.g., diabetes, Date Recue/Date Received 2021-11-12 =
obesity). Accordingly, an initial screening can be conducted to determine the extent to which food intake is modulated by administration of compounds described herein, and a positive initial screening can be useful in subsequent development of a compound.
[01751 In vitro assays. Without wishing to be bound by any theory or mechanism of action, it is believed that correlations exist between the results of in vitro (e.g., receptor) assays, and the utility of agents for the treaunent of metabolic diseases and disorders.
Accordingly, in vitro assays (e.g., cell based assays) are useful as a screening strategy for potential metabolic agents, such as described herein. A variety of in vitro assays are known in the art, including those.
described as follows.
101761 Leptin binding assay; Leptin binding can be measured by the potency of a test compound in displacing .1.231-recombinant-Leptin (murine) from the surface membrane expressing chimeric Leptin (Hu) ¨ EPO (Mu) receptor presented by the32D OBECA
cell line CI -Bird Chem 1998; 273(29): 18365-18373). Purified cell membranes can be prepared by homogenization from harvested confluent cell cultures of 32D OBECA cells.
Membranes can be incubated- with 121I-rec-/vturine-Leptin and increasing concentrations of test compound for 3 hours at ambient temperature in 96-well polystyrene plates. Bound and unbound ligand fractions can then be separated by rapid filtration onto 96-well GF/B plates pm-blocked for at least 60' in 0.5% PEI (polyethyleneimine). Glass fiber plates can then be dried, scintillant added, and CPM
determined by reading on a multiwell scintillation counter capable of reading radiolabeled iodine.
101771 Leptin functional assay. Increased levels of phosphorylated STAT5 (Signal Transducer and Activator of Transcription 5) can be measured following treatment of 32D-Keptin cells ectopicaily expressing chimeric Hu-Leptiniltilu-EPO receptor with a test compound.
The 32D-Keptin cells (identical to 32D-OBECA cells but maintained in culture with [coin) can be leptin weaned overnight and then treated with test compounds in 96-well plates for 30 minutes at 37 C followed by cell extraction. The pSTAT5 levels in the cell lysates can be determined using the 'Perkin Elmer AlphaSereen Surefire pSTAT5 assay kit in a 384-well format (ProxiplatcTM 384 Plus). Thc efficacy of test compounds can be determined relative to the maximal signal in cell lysatcs from cells treated with Human leptin.

Date Recue/Date Received 2021-11-12 VI. Pharmaceutical Compositions 101781 In one aspect, there are provided pharmaceutical compositions comprising compounds described herein in combination with a pharmaceutically acceptable excipient (e.g., carrier). The term "pharmaceutically acceptable carrier," as used herein refers to pharmaceutical excipients, for example, pharmaceutically, physiologically, acceptable organic or inorganic carrier substances suitable for enteral or parenteral application that do not deleteriously react. with the active agent. Suitable pharmaceutically acceptable carriers include water, salt solutions (e.g., Ringer's solution and the like), alcohols, oils, gelatins, and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxyrnethycellulose. and polyvinyl pyrrolidine. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, =
= preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substatices and the like that do not deleteriously react with the compounds of the invention.
=
A. Methods 101701 The chimeric polypeptides- described herein can be administered alone or can be co-administered to .a subject. Co-administration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). For example, it has been found that obesity can be beneficially treated with a combination therapy =
including a leptin (e.g., metreleptin) and certain other anti-obesity compounds. See e.g., U.S.
Published App!. No. 2008/0207512. Accordingly, a chimeric polypeptide described herein could be useful for treatment of obesity.
[01.801 In some embodiments, the formulations and methods described herein further provide that the chimeric polypeptide is co-administered with one or more anti-diabetic agents, such as anti-hyperglycemia agents, e.g. insulin, amylins, prandintide, metfomin.
101811 In some embodiments, the formulations and methods described herein further provide that the chimeric polypeplide is co-administered with one or more cholesterol anclior triglyceride lowering agents. Exemplary agents include ITMG CoA reductase inhibitors (e.g., atorvastatin, =
fluvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin); bile ace sequestrants (e.g., colescvelam, cholestyramine, colestipol): fibrates (e.g., fenofibrate, clofibrate, gemlibrozil);
= =

Date Recue/Date Received 2021-11-12 ezetimibe, nicotinic acid, probucol, a lovastatininiacin combination; an atorvastatin/amlodipine combination; and a simvastatinlezetimibe combination.
[01821 Alternatively, the individual chimeric polypeptides can be co-administered with other anti-obesity agents, such as exenatide or liraglutide.
[01831 The present disclosure provides the composition for use as a medicament, i.e. for use in therapy, since the leptin compound is a therapeutically active compound.
Compositions comprising a chimeric poly-peptide, either liquid or dry form, and optionally at least one pharmaceutically acceptable carrier and/or excipient are also specifically contemplated and are exemplified herein.
[01841 . Co-administration can he achieved by separately administering the chimeric polypeptide with the second agent, or by administering a single Pharmaceutical formulation comprising the chimeric polypeptide and the second agent. Appropriate dosage regimens for the second agents are generally known in the art.
[01851 The preparations can also be co-administered, when desired, with other active substances (e.g. to reduce metabolic degradation) as known in the art or other therapeutically active agents.
101861 Amytins. Amylin is a peptide hormone synthesized by pancreatic 11-cells that is co-secreted with insulin in response to nutrient intake. The sequence of amylin is highly preserved across mammalian species, with structural similarities to calcitonin gene-related peptide (CGRP), the calcitonins, the intermedins, and adrenornedullin, as known in the an. The glucoregulatory = actions of amylin complement those of insulin by regulating the rate of glucose appearance in the circulation via suppression of nutrient-stimulated glucagon secretion and slowing gastric emptying. In insulin-trcated patients with diabetes, pramlinticie, a synthetic and equipotent , analogue of human ainylin, reduces postprandial glucose excursions by suppressing inappropriately elevated postprandial gincagon secretion and slowing gastric emptying.
The sequences of rat amylin, human amylin and nramlintide follow:
rat ainylin: KCNTATCATQRLANTIVIZSSNNLGPVLITINVGSNTY (SEQ 11) NO:86);
human arnylin: KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (SEQ ID NO:87), Pramlintide: KCNIATCATQRLANFINHSSNNFGPELPPINVGSNTY(SEQ ID NO:88).

=
Date Recue/Date Received 2021-11-12 (0187) Davalintide. Davalintide, also known as "AC-2307" is a potent amylin agonist useful in the treatment of a variety of disease indications. See WO 2006/083254 and WO 2007/114838.
Davalintide is a chimeric peptide, having an N-terminal loop region of amylin or caleitonin and analogs thereof, an alpha-helical region of at least a portion of an alpha-helical region of calcitonin or analogs thereof or an alpha-helical region having a portion of an amylin alpha-helical region and .
a calcitonin alpha-helical region or analog thereof, and a C-terminal tail region of amylin or calcitonin. The sequences of human calcitonin, salmon calcitonin and davalintide follow:
human calcitonin: CONLSTCM.I.OTYTQINNKFTITFPQTAIOVGA.P (SEQ ID NO:89);
salmon calcitonin: CSNLSTCVLOKLSQELHKLQTY PRTNTGSGIP (SEQ ID NO:90):
davalintide: KCNTATCVLGRLSQELHR.LQTYPRTNICSNTY (SEQ. ID NO:91).
[01881 Without wishing to be bound by any theory, it is believed that antylins and davalintide, and fragments and analogs thereof, can require C-terminal amidation to elicit a full biological response. It is understood that amylin compounds such as those described herein which include amylins and/or davalintide, and fragment and analogs thereof, can be ainidated at the C-terminal.
[01891 "Amylin agonist compounds" include native antylin peptides, arnylin analog peptides, and other compounds (e.g., small molecules) that have amylin agonist activity.
The "amylin agonist compounds" can be derived from natural sources, can be synthetic, or can he derived from recombinant DNA techniques. Amylin agonist compounds have amylin agonist receptor binding activity and may comprise amino acids (e.gõ natural, unnatural, or a combination thereof), peptide mimeties, chemical moieties, and the like. The skilled artisan will recognize amylin agonist compounds using amylin receptor binding assays or by measuring arnylin agonist activity in soleus muscle assays. In one embodiment, amylin agonist compounds will have an IC50 of about 200 nM or less, about 100 nM or less, or about 50 nM or less, in an amylin receptor binding assay, such as that described herein, in US Patent No. 5,686,411, and US Publication No. 2008/0176804.
In one embodiment, amy I in agonist compounds will have an ECso ulabout 20 nM or less, about 15 nM or less, about 10 rIM or less, or about 5 nM or less in a soleus muscle assay, such as that described herein and in US Patent No.
5,686,411. in one embodiment, the arnylin agonist compound has at least 90% or 100% sequence identity to =
Date Recue/Date Received 2021-11-12 25'2329Pro-human-amvlin. In one embodiment, the amylin agonist compound is a peptide chimera of amylin. (e.g., human am.ylin, rat amylin, and the like) and calcitonin (e.g., human calcitonin, salmon calcitonin, and the like). Suitable and exemplary amylin agonist compounds are also described in US Publication No. 2008/0274952.
101901 By "atnylin analog" as used herein is meant an arnylin agonist that has at least 50%
sequence identity, preferably at least 70% sequence identity, to a naturally-occurring form of amylin, either rat or human or from any other species, and is derived from them by modifications including insertions, substitutions, extensions, and/or deletions of the reference amino acid sequence.
19191,1 The amylin analog sequence can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% amino acid sequence identity with the reference arnylin. En one aspect the analog has 0, I, 2, 3, 4, 5, 6, 7, 8, 9, 10,1.1. 12, 13, .14, 15 or even 16 amino acid substitutions, insertions, extensions, and/or deletions relative to the reference compound.
In one embodiment, the amylin analog may comprise conservative or non-conservative amino acid substitutions (including non-natural amino acids and L and D forms). These analogs are preferably peptides, peptide derivatives or peptide mimics. Typical amylin analogs will be peptides, especially of 32-37 amino acids, e.g. 27 1o45, especially 28 to 38, and even 31-36.
101921 Amylin analogs with identity to rat and human amylin include 2"8.29Pro-h-amylin (pramlintide) (SEQ ID NO: 88); des-iLys-h-arnylin (SEQ. ID NO: III);
25Pro,16\41,2"Pro-h-amylin (SEQ ID NO: 112); I8Arg,25'28Pro-h-arnylin (SEQ ID NO: 113); des-I
Lys,18Arg,2528Pro-h amylin (SEQ ID NO: 114); J8Arg,25'28.29Pro-h-arny1in (SEQ ID NO: 115); des-taArg,25,28.29pro_h_amylin ILys, (SEQ ID NO: .116); des-I,Lys2'28.29Pro-h-amylin (SEQ ID
NO:
117); 25Pro,26Va1,18:2911ro-h-amy1in (SEQ ID NO: 118); 28Pro-b-amylin, 2,7-Cyc10-rAsp:Lys)-h-amylin (SEQ ID NO: 119); 2-37h-amy1in (SEQ ID NO: 120); lAla-h-amylin (SEQ ID
NO: 121);
2Ala-h-arnylin (SEQ ID NO: 122); 2.7Ala-h-amylin (SEQ ID NO: .123); 1Ser-h-amylin (SEQ ID
NO: 124); 29Pro-h-amylin (SEQ IT) NO: 125); 2528Pro-h-amylin (SEQ ID NO: 126);
des-I Lys,25'78Pro-h-amylin (SEQ 'ID NO: 127); 23Leu,25Pro,26Val,229Pro-h-amy1in (SEQ ID NO:
128); 23Leu2sPro26Va12sPro-h-amylin (SEQ ID NO: 129);
dcs)Lys,23Leu,a5Pro,26Val,26Pro-h-amy1in (SEQ ID NO: 130); I8Arg,131..eu,25Pro,26Val,23Pro-h-ainylin (SEQ VD NO:
131);

Date Recue/Date Received 2021-11-12 = "Arg,33Leti,25,2s,29Pro-h-amylin (SEQ ID-NO: 132); '"Arg23Leu,252Pro-h-amylin (SEQ ID NO:
133); '71.1e,23Leu,25'28'29Pro-h-amylin (SEQ ID NO: 134); 17:Ile,25'2Pro-h-arnylin (SEQ ID NO:
= 135); desiLys,i'1le,23Leu,"2*-29Pro-h-amylin (SEQ JD NO: 136);
Pl1e,thArg,731,eu-h-amylin (SEQ ID NO: 137); '7Ile,'8Arg,23Lett,21'Val,29Pro-h-amylin (SEQ ID NO:
138);
"I1e,Arg,23Leu,25Pro,26Val,429Pro-h-arnylin (SEQ ID NO: 139);
1;21 flis,23Leu,26Ala,"Leu,29Pro,)1Asp-h-amylin (SEQ ID NO: 140);
'3111r,2'His,23Leu,26A1a,29Pr0,3'Asp-h-urnylin (SEQ 1D NO: 141); des-'Lys,13Thr,2'His,l'Leu,"Ala,"Pro,3'Asp-h-amylin (SEQ ID NO: 142);
'3Thr,"Arg,2' Iiis,231..ett,26Ala,29Pro,31Asp-h-amy1 in (SEQ ID NO: 143); =
umr,isArg,21 His,21Leup2s,29pro,31 sp-b-arnylin (SEQ ID NO: 144); and 3Thr,t'Arg,2'His!3Leu,25pro,26A1332 sp-ixs,29proi3i. h-amylin (SEQ ID NO: 145).
[01931 Amyl in analogs include amino acid sequences of residues 1-37 of Formula (I) following, wherein up to 25% of the amino acids set forth in Formula (1) may be deleted or substituted with a different amino acid:
X '-Xaal-Cys2-Asn3-Thr4-Ala5.-Thr4-Cys7-Ala'-Thr9-Gln` -Argi j-Leu.12-Alau-Asn14-Phe"-Leu"-Val"-Hie-Ser"-Scr2 - Xaa.2' -=
Asn"-Phe"- .Xaa24- Xaa"- Xaa27- Xaa"- Xaa29-Thr30-Xaa3'-Va132-G1y"-Ser34-Ase-Thr36-Tyr37-X (SEQ ID NO:92) = (I).
In Formula (I), X' is hydrogen, an N-terminal capping group, or a linker to a duration enhancing moiety. Xaa' is Lys or a bond, Xaan is Lys, Cys, or Asti, Xaa24 is Lys, Cys, or Gly, Xaa23 is Lys, Cys, or Pro, Xaa"6 is Lys, Cys, or He, .Xakty is Lys, Cys, or Leo, Xaa"
is Lys. Cys, or Fro, =Xaa29 is Lys, Cys, or Pro and Xas" is Lys, Cys, or Asn. Further regarding Formula (1), the variable X represents a C-terminal functionality (e.g., a C-terminal cap). .X
is substituted or unsubstituted amino, substituted or unsubstituted alkylamino, substituted or unsubstituted dialkylamino, substituted or unsubstituted cycloalkylamino, substituted or unsubstituted arylamino, substituted or tuisubstituteci aralkylamino, substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, substituted or unsubstituted aralkyloxy, or hydroxyl. If the C-terminal of the polypeptide component with the sequence of residues 1-37 of Formula (I) is capped with a functionality X, then X. is preferably amine thereby forming a C-terminal amide.
In some embodiments, up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or even 50% of Date Recue/Date Received 2021-11-12 the amino acids of residues 1-37 of Formula (1) are deleted or substituted in a polypeptide component according to Formula (1). In some embodiments, the amylin analog component has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,=11, 12, 13,14, 15 or even 16 amino acid substitutions relative. to the amino acid sequence set forth in Formula (1).,In some embodiments, the amylin analog has n sequence which has a defined sequence identity with respect to the residues 1-37 of the amino acid sequence according to Formula (I). In some embodiments, the sequence identity between an amylin analgo described herein and residues 1-37 of Formula (I) is 50%, 55%, 60%, 63%, 70%, 75%, 80%, 85%, 90%, 95% or even higher. In some embodiments, up to 50%, 45%, 40%, 35%, 30%, 25%, 20%, .15%, 10%, 5% or even less of the amino acids set forth in residues 1-37 or Formula (1) may be deleted or substituted with a different amino acid. In sortie embodiments, the sequence identity is within the range 75%-100%. in some embodiments, the sequence identity is =
= within the range 75%-90%. I:n some embodiments, the sequence identity is within the range 80%-90%. In sonic embodiments, the sequence identity is at least 75%. In some embodiments, =
the amylin analog has the sequence of residues 1.37 of Formula (0.
= 101941 in some embodiments, amylin analogs including those of Formula (I), form the basis of a polypeptide component to which one or more duration enhancing moieties are linked, optionally through a linker, to form an amylin polypeptide conjugate. Thus, the polypeptide component serves as a template ('polypeptide template") to 'which is attached, preferably by covalent attachment, one or more duration enhancing moieties. Linkage of the duration enhancing moiety to the polypeptide component can be through a linker as described herein.
Alternatively, linkage of the duration enhancing moiety to the polypeptide component can be via a direct covalent bond. The duration enhancing moiety can be a water soluble polymer as described herein. In some embodiments, a plurality of duration enhancing moieties are attached to the polypeptide component, in which case each linker to each duration enhancing moiety is independently selected from the linkers described herein.
01951 Antylin analogs useful as polypeptide components described herein include, but arc not =
limited to, the compounds set forth in residues 1-37 of Formula (I) provided in Table 1 below.
Unless indicated to the contrary, all peptides described herein, including peptides having an expressly provided sequence, are contemplated in both fret carboxylate and amidated forms.

=
Date Recue/Date Received 2021-11-12 Table I. Component polypeptides useful in the compounds described herein.
Cmpd Description (sequence) 1 KCNTATCATQRLANFLVRSSNNLGPVI,PTNVGSNTY-NHa(SEQ ID
NO: 110) 2 CITEATCATQRLANFLVRSSNNLOPVI,PPINVOSNTY-NH2(SEQ ID
NO:93) ((desLysli-Crepd I) 3 . KCNTATCATQRLANFLVII.SSKNLGPVI,PPTNVOSNTY-WH2(SEQ ID
NO:94) 4 CNTATCATQRLA NELVItSSKN LCIPVLPPTNVOSNTY-NH 2 (SEQ ID
NO:95) =
([dcslysII-Crepd ]) KC.NTATCA1QR.LA2'FLVRSSNNLOPKOPTNVGSNTY-N12(SEQ ID
NO:96) 6 - CNTATCATQRLANELVRSSNNLGPICLP rrNvoSIITY -N H (SEQ U) NO:97) adesLy.s11-Cmpd 5) 7 KCNTAICATQRLANIIVRSSNNLO PVLYPTKVOSNT Y-N (SEQ ID
.NO:98) ___________________________________________________________ , .
8 ONTATCATQRLANFI.NRSSNNEGPVLPPTKVGSNTY -N H7(SEQ ID
NO:99) (jdesLysll-CnIpd 7) 9 KCNTATCATQUANELVIISSNNEGP11.PPTNVGSNITY-N112(SEQ ID
NO:100) ' CNTATCATQRIANELVIISSNINFGPII.PPINVOSNTY-Nlli(SEQ ID
NO:101) (LcicsLyslI-Ctripd 9)
11 CNIATCATQRLANELVHSSKNEGYILYPINVGSNTY-NH2(SEQ11) NO:102)
12 CNTAICATQUANFLVIISSNNECIPKLPPINVOSN1Y-N11 (SEQ ID
I NO:103)
13 I CNTAICATQRLANFLVHSSNNEGPILPPIKVG SNTY-NH2 (SEQ ID
NO:104)
14 1 CNTATCATQRLANFLVEISSNNTKPILPPT.NVOSNTY-NH2 (SEQ ID
' .NO:105) CNITATCATQRLANFLVHSSNNF(,KILYPTI4VOSNTY-NH.(SEQ .11) NO:106) 16 CNTATCATQRLANFLVHSSIINRIPIKPPINVOSNITY-NH.(SEQ ID
NO:107) 17 CNrTATCATQRLANELVHSSNNFOPILKPINV(iSNIY-N (SEQ ID

Date Recue/Date Received 2021-11-12 Cmpd Description (sequence) 1 NO:108) CNTAICATORLANTINTISSNNTGP11,PKTNNDSNTY-N112(SEO ID
I NO:109) 101961 The terms "linker" and the like, in the context of attachment of duration enhancing moieties to a poly-peptide component in an amylinpolypeptide conjugate described herein;
means a divalent species (-L.-) covalently bonded in turn to a polypeptide componenl having a valency available for bonding and to a duration enhancing moiety having a valency available for bonding. The available bonding site on the polypeptide component is conveniently a side chain residue (e.g., lysine, cysteine, aspartic acid, and hoinologs thereof). In some embodiments, the available bonding site on the polypeptide component is the side chain of a lysine or a cysteine residue. In some embodiments, the available bonding site on the polypepticie component is the N-terminal amine. In some embodiments, the available bonding site on the poly-peptide component. is the C-terminal carboxyl. In some embodiments, the available bonding site on the polypeptide component is a backbone atom thereof. As used herein, the term "linking amino acid residue" means an amino acid within residues I-37 of Formula (I) to which 3 duration enhancing moiety is attached, optionally through a linker. =
= 101971 in some embodiments, compounds are provided having a linker covalently linking a =
polypeptide component with a duration enhancing moiety. The linker is optional; i.e., any linker may simply be a bond. In some embodiments, the linker is attached at a side chain of the =
polypeptide component. In some embodiments, the linker is attached to a backbone atom of the polypeptide component.
[0198] - In another aspect, there is provided an amylin polypeptide conjugate, which is a derivative of pramlintide with SEQ ID NO:88 or an analog thereof, wherein the amino acid residue in position I is absent (i.e., des-Lys') and an amino acid residue in position 2 to 37 has.
been substituted with a lysine residue or cysteine residue and wherein said lysine residue or cysteine residue is linked to a polyethylene glycol polymer, optionally via a linker, wherein the amino acid numbering conforms with the amino acid number in SEQ ID NO:88.
101991 In another aspect, the invention relates to an amylin polypeptide conjugate, which is 3 derivative of pramlimide with SEQ ID NO:88 or an analog thereof, wherein the amino acid =
Date Recue/Date Received 2021-11-12 =
. residue in position 1 is absent (i.e., des-Lys) and wherein an amino acid residue in any one of position 2, 3, 4, 5,6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, IS, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 31, 32, 33, 34, 35, 36, or 37 is substituted with a lysine residue and wherein said lysine residue is linked to a polyethylene glycol polymer, optionally via a linker.
102001 In another. aspect, the invention relates to an amylin polypeptide conjugate, which is a derivative of pramlintide with SEQ ED NO:88 or an analog thereof, wherein the amino acid residue in position 1 is absent (i.e., des-Lys) and wherein an amino acid residue in any one of position 21, 24-29, or 31 is substituted with a lysine residue and wherein said lysine residue is linked to a polyethylene glycol polymer, optionally via a linker.
1102011 In another aspect, the invention relates to an amylin polypeptide conjugate, which is a derivative of ptamlintide with SEQ ED NO:88 or an analog thereof, wherein the amino acid residue in position 1 is absent (i.e., des-Lys) and wherein an amino acid residue in position 21 is substituted with a lysine residue and wherein said lysine residue is linked to a polyethylene glycol polymer, optionally via a linker.
10202-1 In another aspect, the invention relates to an amylin polypeptide conjugate, which is a derivative of pramlintide with SEQ ID NO:88 or an analog thereof, wherein the amino acid residue in position 1 is absent (i.e., des-Lys') and wherein an amino acid residue in position 24 is substituted with a lysine residue and wherein said lysine residue is linked to a polyethylene glycol polymer, optionally via a linker.
102031 En another aspect, the invention relates to an amylin polypeptide conjugate, which is a derivative of prarnlintide with SEQ ID NO:88 or an analog thereof, wherein the amine acid residue in position 1 is absent (i.e., des-Lys) and wherein an amino acid residue in position 25 is substituted with a lysine residue and wherein said lysine residue is linked to a polyethylene glycol polymer, optionally via a linker.
102041 In another aspect, the invention relates to an amylin polypeptide conjugate, which is a derivative of pramlintide with SEQ ID NO:88 or an analog thereof, wherein the amino acid residue in position I is absent (i.e., des-Lys') and wherein an amino acid residue in position 26 is substituted with a Ivsine residue and wherein said lysine residue is linked to a polyethylene = glycol polymer, optionally via a linker.

Date Recue/Date Received 2021-11-12 102051 In another aspect, the invention relates to an amylin polypeptide conjugate, which is a derivative of prarnlintide with SEQ ID NO:88 or an analog thereof, wherein the amino' acid.
residue in position I is absent (i.e., des-Lys') and wherein an amino acid residue in position 27 is .
substituted with a lysinc residue and wherein said lysine residue is linked to a polyethylene glycol polymer, optionally via a linker.
102061 In another aspect, the invention relates to an arnylin polypeptitie conjugate, which is a derivative of pnunlintide with SEQ ID NO:88 or an analog thereof, wherein the amino acid residue in position I is absent (i.e., des-Lys') and wherein an amino acid residue in position 28 is substituted with a lysine residue and wherein said lysine residue is linked to a polyethylene glycol polymer, optionally via a linker. =
102071 In another aspect, the invention relates to an amylin polypeptide conjugate, which is a derivative of pramlintide with SEQ ID. NO:88 or art analog thereof, wherein the amino acid residue in position 1 is absent (i.e., des-Lys') and wherein an amino acid residue in position 29 is substituted with a lysine residue and wherein said lysine residue is linked to a polyethylene glycol polymer, optionally via a linker.
102081 In another aspect, the invention relates to an ainylin polypeptide conjugate, which is a =
derivative of prarnlintide with SEQ ED NO:88 or an analog thereof, wherein the amino acid residue in position 1 is absent (i.e., des-Lys') and wherein an amino acid residue in position 31 is substituted with a lysine residue and wherein said lysine residue is linked to a polyethylene glycol polymer, optionally via a linker.
102091 In some embodiments, the duration enhancing moiety is a water-soluble polymer. A
'.`water soluble polymer' means a polymer which is sufficiently soluble in water under physiologic conditions of e.g., temperature, ionic concentration and the like, as known in the art, =
to be useful for the methods described herein. A water soluble polymer can increase the solubility of a peptide or other biomolecule to which such water soluble polymer is attached.
Indeed, such attachment has been proposed as a means for improving the circulating life, water solubility and/or antigenicity of administered proteins, in vivo. See e.g., U.S. Pat. No. 4,179,337;
U.S. Published Appl. No, 2008/0032408. Many different water-soluble polymers and attachment chemistries have been used towards this goal, such as polyethylene glycol, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl =
Date Recue/Date Received 2021-11-12 pyrrolidone,"poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylenemaleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and the like.
102101 In some embodiments, the linked duration enhancing moiety includes-a polyethylene glycol. Polyethylene glycol ("PEG") has been used in efforts to obtain therapeutically .usable polypeptides. See e.g., Zalipsky, S., 1995, Bioconjugate Chetnisny, 6:150-165;
Mehvar, R., 2000,). Pharni. Pharmaceut Sd., 3:125-136. As appreciated by one of skill in the art, the PEG -. backbone [(CH2CH2-0-),, n: number of repeating monomers] is flexible and amphiphilic.
Without wishing lobe bound by any theory or mechanism of action, the long, chain-like PEG
molecule or moiety is believed to be heavily hydrated and in rapid motion when in an aqueous medium: This rapid motion is believed to cause the PEG to sweep out a large volume and prevents the approach and interference of other molecules. As a result, when attached to another chemical entity (such as a peptide), PEG polymer chains can protect such chemical entity front .
immune response and other clearance mechanisms. As a result, pegylation can lead to improved drug efficacy and safety by optimizing pharrnacokineties, increasing bioavailability, and decreasing immunogenicity and dosing frequency. "Pegylation" refers in the customary sense to conjugation of a PEG moiety with another compound. For example, attachment Of PEG has been shown to protect proteins against proteolysis. See e.g., illomhoff, H. K.
et al., 1983, Binchirn Binphys Acta, 757:202-208. Unless expressly indicated to the contrary, the terms "PEG, " "polyethylene glycol polymer" and the like refer to polyethylene glycol polymer and derivatives thereof, including methoxy-PEG (mPEG).
102111 A variety of means have been used to attach polymer moieties such as PEG and related polymers to reactive groups found on the protein. See e.g., U.S. Pat. No.
4,179,337; U.S. Pat.
No. 4,002,531; Abuchowski etal., 1981, in " 'Enzymes as Drugs," J. S.
Holeerberg and J. Roberts, (Eds.), pp.. 367-383; Zalipsky, S., 1995, Bioconjugare Chemistry, 6:150-165.
The use of PEG
and other polyinem to modify proteins has been discussed. See e.g., Cheng, T.-L, etal.. 1999m, Bioconjugate Chem., 10:520-528; Belcheva, N. et al.,1999, Bioconjugate Chem., 10:932-937;
Bettinger, T. etal., 1998, Bioconjugate Chem., 9:842-846; Huang, S.-Y. etal., 1998, Bioconjugate Chem., 9:612-617; Xu, B. et al. 1998, Langmuir, 13:2447-2456;
Schwarz, J. B. et -al., 1999, J. Amer. Chem. Soc., 121:2662-2673; Reuter, J. D. eta!,, 1999, Biacanjugate Chem., 10:271-278; Chan, T.-H. etal.. 1997,). Org. Chem., 62:3500-3504, Typical attachment sites in =
Date Recue/Date Received 2021-11-12 proteins include primary amino groups, such as those on lysine residues or at the N-terminus, thiol groups, such as those on cysteine side-chains, and carboxyl groups, such as those on glutamate or aspartate residues or at the C-terminus. Common sites of attachment are to the sugar residues of glycoproteins, cysteines or to the N-terminus and lysincs of the target polypeptide. The terms "pegylated" and the like refer to covalent attachment of polyethylene glycol to a polypeptide or other biomolecule, optionally through a linker as described herein and/or as known in the art.
102121 In some embodiments, a PEG moiety in an amylin polypeptide conjugate described = herein has a nominal molecular weight within a specified range. As customary in the art, the size or a PEG moiety is indicated by reference to the nominal molecular weight, typically provided in kilodaltons (kI21). The molecular weight is calculated in a variety of ways known in the art, including number, weight, viscosity and "Z" average molecular 'weight. It is understood that polymers, such as PEG and the like, exist as a distribution of molecule weights about a nominal average value.
102131 Exemplary of the terminology for molecular weight for PEGs, the term "inPEG40K.D'' refers to a methoxy polyethylene glycol polymer having a nominal molecular weight of 40 kilodaltons. Reference to PEGs of other molecular weights follows this convention. In some embodiments, the PEG moiety has a nominal molecular weight in the range. 10-100 KD, 20-80 KD, 20-60 KD, or 20-40 KD. In some embodiments, the PEG moiety has a nominal molecular weight of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or even 100 Preferably, the PEG moiety has a molecular weight of 20, 25, 30, 40, 60 or 80 KD, [02141 PEG molecules useful for clerivatization of polypcptides arc typically classified into linear, branched and Warwick (i.e., PolyPEGg) classes of PEGs, as known in the art. Unless expressly indicated to the contrary, the PEG moieties described herein are linear PEGs.
Furthermore, the terms "two arm branched," "Y-shaped" and the like refer to branched PEG
moieties, as known in the art. The term "Warwick" in the context of PEGs, also known as "comb" or "comb-type" PEGs, refers to a variety of multi-arm PEGs attached to a backbone, typically poly(methaerylate), as known in the an. Regarding nomenclature including conventions employed in the table provided herein, absent indication to the contrary a PEG
moiety is attached to the backbone of the peptide. For example, Cmpd 19 is the result of the Date Recue/Date Received 2021-11-12 conjugation of mPEG40KD to the N-terminal nitrogen of Cmpd 1. Similarly, Cmpd 20 is the result of conjugation of mPECI4OKD to the N-terminal nitrogen of Cmpd 2.
Standanl single letter abbreviations for amino acids can be used, as can standard three-letter abbreviations. For example, Cmpd 24 is an analog of Cmpd 10 wherein the residue at position 26.of Cmpd 9 is substituted for lysine, and the pendant amine functionality of lysine 26 (i.e., K26) is conjugated with a PEG4OKD moiety. Exemplary compounds are provided in Table 2 below.
Table 2. Pegylated compounds Cmpd Description 19 inPEG40KD-Cmpd I (SEQ ID NO: 146) 20 mYEG40KD-Cmpd 2 (SEQ ID NO: 147) 21 1le(inYEG4OKD)).Ciopd 3 (SEQ ID NO: 148) 22 11.1.1'1(niP.EG40KI)1-Cmpd 4 (SEQ ID NO: 149) 23 10(n1PE(.140KD)J-Cmpd 5 (SEQ ID NO: 150) 24 (K2 (11nPE640K0))-Cmpd 6 (SEQ ID NO: 151) 25 IK31(ntPEG40K0)1-Cinpd 7 (SW ID NO: 152) 26 110(mPEG401(D)1-Cmpd 8 (SEQ ID NO: 153) 27 IIC16(Y-shaptii-mPE(1401CD)J-Cmpd S (SEQ ID NO: 154) 28 (1{21(inPF.G4OKD)j-Cinpd 11 (SEQ ID NO: 155) 29 (K2'(inPLIG40KD)1-Cmpd 12 (SEQ ID NO: 156) 30 IK3(11-aPEG40KB))-Cinpd 13 (SEQ ID NO: 157) 31 110(Y-shaped-inPEG4OKD)j-Cmpd 12 (SEQ ID NO: 158) 32 (10(mPEC.140K0)].Cmpd 14 (SE() Ill NO:. 159) 33 IK25(InPEG40K.D))-Cmpd 15 (SEQ ID NO: 160) 34 re(mPEG401.(12)1-Cmpd 16 (SEQ ID NO: 161) 35 (0(ral'EG4010)1-Cmpd 17 (SEQ ID NO: I 62) 36 (1(29(mPEG4OKD)1-Cmpd 18 (SEQ ID NO: 163) B. Formulations 102151 The pharmaceutical compounds of the invention may he formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and Date Recue/Date Received 2021-11-12 excipients in accordance with conventional techniques such as those disclosed in Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang et al. (1988)1 of Pareneerat Sei. and Tech., Technical Report No. 10, Supp. 42:2 S.
102161 In general, the chimeric polypeptides may be fommlated into a stable, safe pharmaceutical composition for administration to a patient. Pharmaceutical formulations contemplated for use in the methods of the invention may comprise approximately 0.01 to 1.0% =
(w/v), in certain cases 0.05 to 1.0%, of the chimeric polypeptide, approximately 0.02 to 0.5%
(w/v) of an acetate, Phosphate, citrate or glutamate buffer allowing a pH of the final composition of from about 3.0 to about 7.0; approximately 1.0 to 10$10(w/v) of a carbohydrate or polyhydric alcohol tonicifier and, optionally, approximately 0.005 to 1.0% (wfv) of a preservative selected -from the group consisting of m-cresol, benzyl alcohol, methyl, ethyl, propyl and butyl parabens and phenol. Such a preservative is generally included if the formulated peptide is to be included in a multiple use product.
[02171 In particular embodiments, a pharmaceutical formulation of the present chimeric =
polypeptides may contain a range of concentrations Of the compound(s), e.g., between about 0.01% to about 98% wive, or between about 1 to about 98% w/w, or preferably between 80% and 90% w/w, or preferably between about 0.01% to about 50% w/w, or more preferably between about 10% to about 25% w/w in these embodiments. A sufficient amount of water for injection may be used to obtain the desired concentration of solution.
[02181 Additional tonicifying agents such as sodium chloride, as well as other known excipients, may also be present, if desired. in some cases, such excipients are useful in maintenance of the overall tonicity of the compound. An excipient may be included in the presently described formulations at various concentrations. For example, an excipient may be included in the concentration range from about 0.02% to about 20% w/w, preferably between =
about 1).02% and 0.5% wiw, about 0.02% to about 10% w/v, or about 1% to about 20% w/w. in .
addition, similar to the present formulations themselves, an excipient may be included in solid (including powdered), liquid, .semi-solid or gel form.
102191 The pharmaceutical formulations tnay be composed in various forms.
e.g., solid, liquid, semisolid or liquid. The term "solid", as used herein, is meant to encompass all normal uses of Date Recue/Date Received 2021-11-12 this term including, for example, powders and lyophilized formulations. The presently described formulations may be lyophilized.
102201 The terms buffer, buffer solution and buffered solution, when used with reference to hydrogen-ion concentration or pH, refer to the ability of a system, particularly an aqueous sOlution, to resist a change of pH on adding acid Or alkali, or on dilution with a solvent.
Characteristic of buffered solutions, which undergo small changes of pH on addition of acid or base, is the presence either of a weak acid and a salt of the weak acid, or a weak base and a salt of the weak base. An example of the former system is acetic acid and sodium acetate. The = change-01pH is slight as long as the amount of hydronium or hydroxyl ion added does not exceed the capacity of the buffer system to neutralize it.
10221.1 As described herein, a variety of liquid vehicles are suitable for use in the formulations of chimeric polypeptides, for example, water or an aqucouslorganic solvent mixture or =
suspension.
102221 The stability of a chimeric polypeptide formulation for use as described herein is = enhanced by maintaining the pH of the formulation in a range determined by methods known in . the art. lri certain embodiments, the pH of the formulation is maintained in the range of about 3.5 to 5.0, or about 3.5 to 6.5, in some embodiments from about 3.7 to 4.3, or about 3.8 to 4.2. In some embodiments, pH may be about 4.0, about 5.0, about 6,0, about 7.0, about 8.0, about 9.0, or even higher. In some embodiments, pH may be in the physiological range, pH
6-8, preferably pH 7-7.6.
102231 In certain embodiments, the buffer with the chimeric potypeptide is an acetate buffer (preferably at a final formulation concentration of from about 1-5 to about 60 inM), phosphate buffer (preferably at a final formulation concentration of from about. 1-5 to about to about 30 nilvt) or glutamate buffer (preferably at a final formulation concentration of from about 1-5 to about to about 60 mM). In some embodiments, the buffer is acetate (preferably at a final .
= formulation concentration of li-om about 5 to about 30 nil%
. 102241 A stabilizer may be included in the formulations but is not necessarily needed. If included,. however, a stabilizer useful in the practice of the present invention is a carbohydrate or a polyhydric alcohol. A suitable stabilizer useful in the practice of the present invention is =
Date Recue/Date Received 2021-11-12 approximately 1.0 to 10% (w/v) of a carbohydrate or polyhydric alcohol. The polyhydric alcohols and carbohydrates share the same feature in their backbones.
which is responsible for stabilizing the pmteins. The polyhydric alcohols include such compounds as sorbitol, mannitol, glycerol, and polyethylene glycols (PEGs).
These compounds are straight-chain molecules. The carbohydrates, such as tnannose, ribose, sucrose, fructose, trehalose, maltose, inositol, and lactose, on the other hand, are cyclic molecules that may contain a keto or aldehyde group. These two classes of compounds have been demonstrated to be effective in stabilizing protein against denaturation caused by elevated temperature and by freeze-thaw or freeze-drying processes. Suitable carbohydrates include:
galactose, arabinose, =
lactose or any other carbohydrate which does not have an adverse affect on a diabetic patient, i.e., the carbohydrate is not metabolized to form unacceptably large concentrations of glucose in the blood. Such carbohydrates are well known in the art as suitable for diabetics. Sucmse and fructose am suitable for use with the compound in non-diabetic applications (e.g. treating obesity).
102251 In certain embodiments, if a stabilizer is included, the compound is stabilized with a polyhydric alcohol such as sorbitol, mannitol, inositol, glycerol, xylitol, and polypropylene/ethylene glycol copolymer, as well as various polyethylene glycols (PEG) of molecular weight 200, 400, 1450, 3350, 4000,6000, 8000 and even higher).
Mannitol is the preferred polyhydric alcohol in some embodiments. Another useful feature of the lyophilized formulations of the present invention is the maintenance of the tonicity of the lyophilized formulations described herein with the same formulation component that serves to maintain their stability. In some embodiments, mannitol is the preferred polyhydric alcohol used for this purpose.
102261 The United States Pharmacopeia (USP) States that anti-microbial agents in bacleriostatic or fungistatic concentrations must be added to preparations contained in multiple dose containers. They must be present in adequate concentration al. the lime of use to prevent the multiplication of microorganisms inadvertently introduced into the preparation while withdrawing a portion of the contents with a hypodermic needle and syringe, or using other invasive means for delivery, such as pen injectors. Antimicrobial agents should be evaluated to ensure compatibility with all other components of the formula, and their activity should be = 78 Date Recue/Date Received 2021-11-12 =
=
evaluated in the total formula to ensure that a particular agent that is effective in one formulation is not ineffective in another. It is not uncommon to find that a particular antimicrobial agent will be effective in one formulation but not effective in another formulation.
102271 A preservative is, in the common pharmaceutical sense, a substance that prevents or inhibits microbial growth and may be added to pharmaceutical tbrmulations for this purpose to avoid consequent spoilage of the formulation by microorganisms. While the amount of the = preservative is not great, it may nevertheless affect the overall stability of the peptide.
102281 While the preservative for use in the pharmaceutical compositions can range from 0.005 to 1.0% (wiv), in some embodiments range for each preservative, alone or in combination with others, is: benzyl alcohol (0.1-1.0%), or m-cresol (0.1-0.63'0, or phenol (0.1-0.8%) or combination of methyl (0,05-0.25%) and ethyl or propyl or butyl (0.005%41.03%) parabens. The parabens are lower alkyl esters of para-hydroxybenzoic acid. A detailed description of each preservative is set forth in Remington's Pharmaceutical Sciences (I)) [0229I Chimeric polypeptities may not have a tendency to adsorb onto the glass in a glass =
container when in a liquid form, therefore, a surfactant may not be required to further stabilize the pharmaceutical formulation, However, with regard to compounds which do have such a tendency when in liquid form, a surfactant should be used in their formulation. These formulations may then be lyophilized. Surfactants frequently cause denaturation of protein, both = of hydrophobic disruption and by salt bridge separation. Relatively low concentrations of surfactant may exert a potent denaturing activity, because of the strong interactions between surfactant moieties and the reactive sites on proteins: However, judicious use of this interaction can stabilize proteins against interfacial or surface denaturation.
Surfactants which could further stabilize the chimeric polypeptide may optionally be present in the range of about 0.001 to 0.3%
(wiv) of the total formulation and include polysorbate 0 (i.e., polyoxyethylenc(20) sorbilan monooleate),'CHAPS (i.e., 3i(3-cholamidopropyl) dimethylammonioll-propanesulfonate), Brij (e.g., Brij 35, which is (polyoxyethylene (23) lauryi ether), poloxamer, or another non-ionic surfactant, 102301 It may also be desirable to add sodium chloride or other salt to adjust the tonicity of the pharmaceutical formulation, depending on the tonicifier selected. However, this is optional and =
Date Recue/Date Received 2021-11-12 =
=
depends on the particular formulation selected. Parenteral formulations preferably may be.
isotonic or substantially isotonic.
(02311 A preferred vehicle for parenleral products is water. Water of suitable quality for parcnteral administration can be prepared either by distillation or by reverse osmosis. Water for injection is the preferred aqueous vehicle for use in the pharmaceutical formulations.
102321 it is possible that other ingredients may be present in the pharmaceutical formulations.
Such additional ingredients may include; e.g., wetting agents, emulsifiers, oils, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g.; human serum albumin, gelatin or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histieline). Additionally, polymer solutions, or mixtures with polymers provide the opportunity for controlled release of the peptide. Such -= additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.
102331 Containers are also an integral part of the formulation of an injection and may be considered a component, for there is no container that is totally inert, or does not in some way affect the liquid it contains, particularly if the liquid is aqueous.
Therefore, the selection of a container for a particular injection must be based on a consideration of the composition of the container, as well as of the solution, and the treatment to which it will be subjected. Adsorption of the peptide to the glass surface of the vial can also be minimized, if necessary, by use of borosilicate glass, for example, Wheaton Type 1 borosilicate glass #33 (Wheaton Type 1-33.) or = its equivalent (Wheaton Glass Co.). Other vendors of similar borosilicate glass vials and cartridges acceptable for mmufacturc include Kimbel Glass Co., West Co., Bander G las GMBH
and Form a Vitruin. The biological and chemical properties of the compound may be stabilized by formulation and lyophilization in a Wheaton Type 1-33 borosilicate semm vial to a final concentration of 0.1-mg/m.I and 10 of the compound in the presence of 5%
mannitol, and 0.02% Tween 80.
102341 For formulations to be delivered by injection, in order to permit introduction of a needle from a hypodermic syringe into a multiple-dose vial and provide for resealing as soon as the needle is withdrawn, the open end of each vial is preferably sealed with a rubber stopper closure held in place by an aluminum band.
ao Date Recue/Date Received 2021-11-12 [02351 Stoppers for glass vials, such as, West 4416150, 4416/50 (TeflonTm faced) and 4406/40, Abbott 5139 or any equivalent stopper can be used as the closure for pharmaceutical for injection. For formulations comprising peptidie anti-obesity agents, these stoppers are compatible with the peptide as well as the-other components of the formulation. Alternatively, the peptide can be lyophilized in to vials, syringes or cartridges for subsequent reconstitution.
Liquid formulations of the present invention can be Filled into one or two chambered cartridges, or one or two chamber syringes.
[0236] Each of the components of the pharmaceutical tbn.nulation described above is known in the art and is described in PHARMACEIMCAL DOSAGE FORMS: PARENrrERAL
MEDIcArioNs, vol. 1, 2nd ed., Avis et al. Ed., Mercel Dekker, New York, N.Y. 1992.
(0237) The manufacturing process for the above liquid formulations generally involves =
compounding, sterile filtration and filling steps. The compounding procedure involves dissolution of ingredients in a specific order (preservative followed by stabilizer/tonicity agents, buffers and peptide) or dissolving at the same time.
102381 Alternative formulations, e.g., non-parenteral, may not require sterilization, However, if sterilization is desired or necessary, any suitable sterilization process can be used in developing the peptide pharmaceutical formulation of the present invention.
Typical sterilization processes include filtration, steam (moist heat), dry heat, gases (e.g., ethylene oxide, formaldehyde, chlorine dioxide,.propylene oxide, beta-propiolacttone, ozone.
chloropicrin, peracetic acid methyl bromide and the like), exposure to a radiation source, and aseptic handling.
. Filtration is the preferred method of sterilization for liquid formulations of the present invention.
The sterile filtration involves filtration through 0.45 urn and 0.22 urn (1 or 2) which may be connected in series. Afier filtration, the solution is filled into appropriate vials or containers.
(02391 in certain embodiments, the chimeric polypeptides described herein are administered peripherally to the subjects. in some embodiments, the liquid pharmaceutical formulations oldie present invention are intended for parentcral administration. Suitable routes of administration include intramuscular, intravenous, subcutaneous, intradermal, intraarticular, intrathecal and the like. In some embodiments, the subcutaneous route of administration is preferred. In certain embodiments, mucosal delivery is also preferred. These routes include, but are not limited to, Date Recue/Date Received 2021-11-12 oral, nasal, sublingual, pulmonary and buccal routes which may include administration of the peptide in liquid, semi-solid Of solid form. For formulations comprising chimeric polypeptides, administration via these routes can require substantially more compound to obtain the desired biological effects due to decreased bioavailability compared to parenterai delivery. In addition, parenteral controlled release delivery can be achieved by forming polymeric microcapsules, . matrices, solutions, implants and devices and administering them parenlerally or by surgical means. Examples of controlled release formulations are described in U.S. Pat.
Nos. 6,368,630, 6,379,704, and 5,766,627. These dosage forms may have a lower bi.oavailability due to entrapment of some Of the peptide in the polymer matrix or device. See e.g., U.S. Pat. Nos. 6,379,704, 6,379,703, and 6,296,812.
[02401 The compounds may be provided in dosage unit .forrn containing an amount of the chimeric .polypeptide that will he effective in one or multiple doses.
[02411 As will be recognized by those in the field, an effective amount of the chimeric polypeptide will vary with, many factors including the age and weight of the subject, the subject's physical condition, the condition to be treated, and other factors known in the art. An effective amount of the chimeric polypeptides will also vary with the particular combination administered, As described herein, administration of the chimeric polypeptides in combination may allow for a reduced amount of any of the administered chimeric polypeptides to be an effective amotint.
C. Effective Dosages 102421 Pharmaceutical compositions provided herein include compositions wherein the active ingredient- is contained in a therapeutically effective amount, i.e., in an amount effective to.
achieve its intended purpose. The actual amount effective for a particular application will depend, inter olio, on the condition being treated. For example, when administered in methods to treat diabetes, such compositions will contain an amount of active ingredient effective to achieve the desired result (e.g. decreasing fasting blood glucose in a subject). When administered in methods to treat obesity, such compositions will contain an amount of active ingredient effective to achieve the desired result (e.g. decrease the body mass).
102431 The dosage and frequency (single or multiple doses) of compound administered can vary depending upon a variety of factors, including route of administration;
size, age, sex, health, Date Recue/Date Received 2021-11-12 = =
body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated (e.g., the disease responsive to compounds described herein); presence of other diseases or other health-related problems; kind of concurrent treatment;
and complications from any disease or treatment regimen. Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of the invention.
. 102441 Therapeutically effective amounts for use in humans may be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that:has been found to be effective in animals. The dosage in humans can be adjusted by monitoring one or more physiological parameters, including but not limited to blood sugar and body mass, and = adjusting the dosage upwards or downwards, as described above and known in the art.
102451 Dosages may be varied depending upon the requirements (tithe patient and the compound being employed. The dose administered to a patient, in the context of the present invention, should be sufficient to affect a beneficial therapeutic response in the patient over time.
The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. In one embodiment of the invention, the dosage range is 0.001% to 10% wlv. In another embodiment, the dosage range is 0.1% to 5% w/v.
102461 However, typical doses may contain from a. lower limit of about 0.1 mg to an tipper limit of about 200 mg of the phannaceutical compound per day. Also contemplated are other dose ranges such as 1 mg to 100 mg of the compound per dose, and 3 rag to 70 mg per dose. The doses per day may be delivered in discrete unit doses, or provided continuously in a 24 hour period or any portion of that the 24 hours.
102471 Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
=
=

=
Date Recue/Date Received 2021-11-12 102481 Utilizing the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be planned that does not cause substantial toxicity and yet is entirely effective to treat the clinical symptoms demonstrated by the particular patient This planning should involve the careful choice of active compound by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred mode of administration, and the toxicity profile of the selected agent.
D. Toxicity 102491 The ratio between toxicity and therapeutic effect for a particular compound is its therapeutic index and can be expressed as the ratio between LD50 (the amount of compound lethal in 50% of the population) and F.D50 (the amount of compound effective in 50% of the population). Compounds that exhibit high therapeutic indices are preferred.
Therapeutic index data obtained from cell culture assays and/or animal studies can be used in tbrmulating a range of dosages for use in humans. The dosage of such compounds preferably lies within a range of plasma concentrations that include the ED 5a with little or no toxicity. The dosage may vary -within this range depending upon the dosage fonn employed and the route of administration utilized. See, e.g. Fingl et al., In: Ton PFLARMACOLOGICAL BASIS OF
TitERAPEtalcS, Ch .1, p.1, 1975. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition and the particular method in which the !compound is used.
=
VII. EXAMPLES
Example 1: Chimeric polypeptide recovery [02501 Protein sequences Were designed and back translated using commercial software to DNA sequence for cloning into an E. coil expression vector. Sequences were either obtained as oligonuelemides and stitched together using 'standard PCR amplification techniques, or they were digested from existing expression constructs using standard restriction enzymes and then ligated back together. Sequences expressing the protein of interest were placed in pET45 with a Ti promoter for inducible expression. After constructs were verified by sequencing, the vector DNA
was purified and transformed into an expression host, typically 131..2 l (DE3). A single colony was = selected to gow.a starter culture in 4 ml LB media for ¨6 his. Glycerol stocks were prepared by Date Recue/Date Received 2021-11-12 adding 100u1 80% glycerol to 900u1 stock and store at -80C. Optionally, 500 ul unindliced sample was retained for gel analysis. A 60 ml culture (magic media) was inoculated using 60W
starter culture in a 125m1 Thompson flask and incubated @ 30C overnight.
Remove 250 ul sample for analysis. Spin down and freeze cell pellet for later processing.
102511 Bacterial cells were harvested and subsequently lysed to isolate inclusion bodies. Since . the protein was present in the inclusion bodies, these Were solubilized and the protein refolded at 4C, Proteins were then separated using size exclusion chromatography until only a single band remained and endotoxin levels were acceptable for in vivo testing. Analytical HPLC, RP-LC-MS
and SDS-PAGE gel were run as quality control measures on the final protein.
Protein was distributed to predetermined aliquots and stored at -80C.
Example 2: Biological and pharmaceutical proper-tics =
102521 As set forth in Table 3 following, chimeric polypeptides described herein have comparable, and some even superior, properties compared with A100 (Compound 37, SEQ ID
NO:24). These properties include biological properties such as leptin binding activity, leptin functional activity, and food intake in mice, and pharmaceutical properties such as solubility in neutral pH.
102531 Exemplary assays for leptin binding activity and leptin functional activity were previously described.
[0254] Food intake activity in mice was tested with the following assay:
C57BI..6 female. mice and their food were weighed daily 3 hours prior to lights out. Immediately after weighing, on days 0, 1, 2 and 3 mice were injected SC with leptin compound or mutant in I
)(PBS. Points represent mean +. sd of n=9 cages (3 mice/cage). The results reported under "Mouse Food Intake" in Table 3 correspond to the vehicle corrected; change in 'A body weight measured after Day 4.
102551 Solubility was measured with the following assay: proteins were concentrated at 4C, spun to remove precipitates, then allowed to equilibrate at room temperature overnight. They were filtered to remove precipitates and then the concentration was determined by measuring absorbance at 01)280 and using the theoretical molar extinction coefficient.
Date Recue/Date Received 2021-11-12 ..
=

Table 3. Biological and pharmaceutical properties of chimeric polypeptides , ___________ ,....õ,,,,,i=z., =--4/
.':.; = :=:-:.='. -= '.=:=:, , ,:',,.., ,::. ' ,:k..;.4M:()=.:'.
:.';:j.('.:',::=. ;::'-'].= ; 7- "... ','NflYil.S.F.f..-. '-..:.
,:?. :'.': ' ..,,=.:, s..:: !!.,.:..,:, : , :_ '':, '. '...: '2i,'::.., µj,'::''. '': :.,;:- :..-.;',='..;:::: =
'''-': (91:14:,!'' :.', :]..',õ..:F''':',,!µ' ..: 4.: :::.= . '. ¨ :, = .... :. ,soiiibiRi411.:.", =.4iiiiptia.f.,ilf!,. ,'..,''',: ...'::, =:':':''' 4a.,.,:'' '::i. :','-().::$.: 16,9^:,3,!3!=?:.:- ''' ti1.4&: .µ"' ''.::' .
','= Nivil!Tirics- "' ' . .,L.
','i'e=.,'.',''''''f-ar's:i'l 'y.:.. ..,, . !'.'..=;.'1,','!=.' ':.=....t.i1.11I-'4III!,'!;'-''..'..-!..:..' S'OTID!.:, :-.,b4,Ii.).,g ,'''''' .-,:r1-III(::[9..9A... i ..,.1;:.--..-,:..-,...,, r'="!,''..,":."i' ,:',-,';,,,! 4., , _:'.., ,..,..:!,....!!: :::::
.5'..,,,..: xe:,..,.,,,,,,",:: <, -..',:.-......,-;,..-.,,-.,,,... ,...,, -,E, ,tv.0) 'Nfa:., ',..,-, ,,.;',.-iii.Nit,.-.,,,....,,,.:....,:..::µ,..,..i.--..=
.''.,.=
0.2aM-24 37 0.8nM 0.02 4.053 3 -.1- - -----18---- 28 i, 1.996 _ 0.019 6.7 35 . - = I
.
=
39 53 ND (>.095 3.1 NI) 40 69 2.412 0.588 0 17 41 32 0.555 = 0.028 1 , 10.R 21 I
1 42 61 , 2.017 0.043 I 8. 4 43 55 3.082 0.054 6.5 ¨T-.
, 44 57 ' 3.542 0.032 . 6.4 20 0.029 10.3 18 45 63 ! 0.527 ' ' I 46 59 1, 0.479 0.042 -0-2% 20 !
;
1 .
= -4% _ 47 71 1 0.788 0.0625 15 1 48 .
________________________________________________________________ ' 33 i 0.036 0.039 4.9 14 I
' 1 1 49 81. I 0.105 0.034 4 3 i , I .
1 50 67 j 0.214 0.022 8.3 5 1 ' 51 65 1 0.119 0.038 3.8 25 r 52 73 i ND 0.044 ND . 19 ND = not determined *These numbers do not necessarily represent the maximum solubility of each compound.
Example 3: Stability of Chimeric Peptides =

..
, -Date Reeue/Date Received 2021-11-12 102561 As set forth in Table 4 following, chimeric polypeptides described herein have comparable, and some even superior, physical stability compared with Al 00 (Compound 37, SEQ 11) NO:24). The compounds were formulated in the following buffer: 10mM
glutamic acid, 2% glycine, 1% sucrose, 0.01% Tween 20, pH 4.25 and stored at 37 C. Samples were pulled at T = 0, 2, 5, 7, and 14 days and tested by visual analysis, reverse phase high performance liquid chromatography (HPLC), UV spectrometry, and dynamic light scattering (DLS). As shown in Table 4, the chimeric polypeptides have comparable or superior purity and potency, compared with Compound 37. =
Table 4. Stability of Chimeric Polypeptides cmpa Potency Remaining* Purity Loss" Colnoted Potency' Visual SizeChange (BLS) 38 1in9irn1 95.3% 1.3% 94.6% Clear 1.0 41 Irrgitn1 92.2% 1.9% 90.4% Clear 111101 88.8% 2.7% 8E1% Clear 1.7 42 inigicril 96.3% 14.9% 314% Clear 1.5 49 1rngint1 94.5% 13.0% 31.51 Clear 1.0 irripril 98.5% 10.7% 37.3% Clear 1.0 *neinialized UV patocy relative lo '-norrnalized RP-HPLC purity relatNe to 1.0 "UV potency - LC purity (total soluble - soluble deo) Example 4: Change in Body Weight After Daily Administration of Chimeric Polypeptide.
[02571 Method. C5713I_6 female mice and their food were weighed daily 3 hours prior to lights out. Inunediately after weighing, on days 0, I, 2 and 3 mice were injected SC with leptin compound or mutant in 1xPBS. Points represent mean sd of rt=4 cages (3 mice/cage). Each group (n=12/group) was assigned to receive one of the following: vehicle; Cmpd 41 at 0.1 mg/kg; Cmpd 41 at 0,3 mg/kg; Cmpd 41 at J mg/kg; Cmpd 41 at 3 mg/kg; Cmpd 41 at 5 mg/kg;
Cmpd 41 at 10 mg/kg. Food intake and change in body weight (1)./0 vehicle corrected) were monitored for 4 days, and the results recorded as shown (Figures IA and 1B).
Points represent mean sd of n--4 cages (3 mice/cage). Administered compounds: Vehicle (filled circle); Cmpd 41 at 0.1 mg/kg (triangle tip down); Cmpd 41 at 0.3 mg/kg (open diamond); Cmpd 41 at I mg/kg Date Recue/Date Received 2021-11-12 (open circle); Cnipd 41 at 3 mg/kg (triangle tip up); Cmpd 41 at 5 mg/kg (star); Cmpd 41 at 10 mg/kg (filled diamond).
102581 Results. As depicted in Figures IA and IB, administration of different doses of the chimeric polypeptide resulted in reduced food intake and body weight relative to the group that received vehicle alone. A dose response is observed in Figure IC.
Example 5: Change in Body Weight After Daily Administration of Chimeric Polypeptide.
102591 Method. C57131.6 female mice and their food were weighed daily 3 hours prior to lights out. Immediately after Weighing, on days 0, 1,2 and 3 mice Were injected SC with leptin . compound or mutant in IxP.BS. Points represent mean ad of n-4 cages (3 mice/cage). Each group (n=12/group) was assigned to receive one of the following: vehicle; Cmpd 42 at 0.1 =
mg/kg; Cmpd 42 at 0.3 mg/kg; Cmpd 42 at I mg/kg; Cmpd 42 at 3 mg/kg; Cmpd 42 at 5 mg/kg;
Cmpd 42 at 10 mg/kg. Food intake and change in body weight (14 vehicle corrected) were monitored for 4 days, and the results recorded as shown (Figures 2A and 2B).
Points represent mean sd of n=4 cages (3 !nice/cage). Administered compounds: Vehicle (filled circle); Cmpd 42 at 0.1 mg/kg (triangle tip up); Cmpd 42 at 0.3 mg/kg (triangle tip up);
Cmpd 42 at 1 mg/kg (tilled square); Cmpd 42 at 3 mg/kg (bar above and below point); Cmpd 42 at 5 mg/kg (star);
Cmpd 42 at 10 nig/kg (bar above point):
102601 Results. As depicted in Figures 2A and 2B, administration of different doses of the chimeric polypeptide resulted in reduced food intake and body weight relative to the group that received vehicle alone. A dose response is observed in Figure 2C.
102611 As shown in Table 5 below, the dose responses measured for chimeric.
polypeptides of the invention arc comparable to the dose responses measured for the seal leptin (Cmpd 38) and human leptin (Cmpd 37) from which the chimeric polypeptides are. derived.
Table 5. Dose responses of chirnerie polypeptides Compound ED50 37 . 0.44- 0.6 mg/kg 38 0.8 mg/kg .
= =

Date Recue/Date Received 2021-11-12 41 0.5 mg/kg 42 1.1 mg/kg =
EMBODIMENTS
[02621 .Additional embodiments of the chimeric polypeptides, method of use thereof, and = pharmaceuticals compositions described herein follow:
102631 Embodiment I. = A chimeric polypeptide comprising a wild type seal leptin polypeptide wherein at least one contiguous region of 1-30 amino acids of a wild type seal leptin sequence has been replaced with a contiguous region of 1-30 amino acids Ca mature human leptin sequence.
102641 Embodiment 2. The chimeric polypeptide according to Embodiment 1, wherein two or more contiguous regions of 1-30 amino acids of a wild type seal leptin sequence have been replaced at each region with a contiguous region of 1-30 amino.acids of a mature human leptin. sequence.
102651 Embodiment 3. The chimeric polypeptidc according to Embodiment I or 2, wherein a wild type seal leptin sequence comprises the amino acid sequence or SEQ ID NO:28 or SEQ ID NO:31.
102661 Embodiment 4. . The chimeric polypeptide according to any one of Embodiments 1-3. wherein a mature human leptin sequence comprises an amino acid sequence selected from the group consisting of: SEQ 1D NO:12, SEQ ID NO:13, SEQ ID .NO:20, SEQ 11) NO:21, SEQ
NO:22, SEQ ID NO:4 SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ

ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID
NO:39, SEQ ID NOAO, SEQ ID NO:41, SEQ ID NO:42, SEQ 1D NO:43, SEQ ED NO:44, SEQ ID
NO:45, SEQ ED NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID 'NO:49, SEQ ID NO:50, and SEQ ID NO:51. =
102671 Embodiment 5. The chimeric polypeptide of any one of Embodiments 1-4, wherein a mature, human leptin sequence comprises an amino acid sequence selected from the group Date Recue/Date Received 2021-11-12 consisting of: SEQ ID NO:12, SEQ ED NO:13, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ II) NO:38, SEQ ED NO:39, SEQ
ID NOAO, SEQ ID NO:41, SEQ ED NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ED NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ 11) NO:50, and SEQ
ID
NO:51, wherein the mature human leptin sequence has at least one amino acid substitution at a position where'divergence is observed in a corresponding position in a leptin from another species. =
[0268] Embodiment 6. The chimeric polypeptide of any one of Embodiments 1-5, wherein a mature binnan leptin sequence comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:12, SEQ ED NO:13, SEQ TD NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ED NO:27, SEQ
ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ
ID NO:40, SEQ ID NO:41, SEQ NO:42, SEQ ID NO:43, SEQ ID N0:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ
ID
NO:51, wherein the mature human leptin sequence has at least two amino acid substitutions at = positions where divergence is observed in corresponding positions in a leptin from another species.
(0269] Embodiment 7. The chimeric polypeptide of any one of Embodiments 1-6, wherein a mature human leptin sequence comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:2 I, SEQ ID
NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ED NO:39, SEQ
ID NOAO, SEQ ID NO:41, SEQ 1.0N0A2, SEQ ID NO:43, SEQ ID NOA4, SEQ ED NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ
ID
NO:51, wherein the mature human leptin sequence has at least three amino acid substitutions at positions where divergence is observed in corresponding positions in a leptin.
from another species.
10270] Embodiment 3. The chimeric polypeptide of any one of Embodiments 1-7, wherein a mature human leptin sequence comprises an amino acid sequence selected from the group =
Date Recue/Date Received 2021-11-12 =
consisting of: SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ 1D
'NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ NO:39, SEQ

ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID
NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID 'NO:49, SEQ ID NO:50, and SEQ
ID
NO:51, wherein the mature human leptin sequence has at least four amino acid substitutions at positions where divergence is observed in corresponding positions in a leptin front another species.
102711 Embodiment 9. The chimeric polypeptide of any one of Embodiments 1-8, wherein a mature human leptin sequence comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID N0:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID 'NO:27, SEQ ID
'NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ED NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ
ID NO:40, SEQ ED NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID 140:44, SEQ ID
NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID 'NO:49, SEQ ID NO:50, and SEQ
ID
NO:51, wherein the mature human leptin sequence has at least five amino acid substitutions at.
positions where divergence is observed in corresponding positions in a leptin from another = species.
(0272! Embodiment 10. The chimeric polypeptide of any one of Embodiments 1-9, wherein a mature human leptin sequence comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:.24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:77, SEQ ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ NO:39, SEQ
-ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID
NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NOA9, SEQ NO:50, and SEQ ID
NO:51, wherein the mature human leptin sequence has at least six amino acid substitutions at positions where divergence is observed in corresponding positions in a leptin front another species.
= [0273j Embodiment 11. The chimeric polypeptidc of any one of Embodiments 1-10, wherein a mature human leptin sequence comprises an amino acid sequence selected from the Date Recue/Date Received 2021-11-12 group consisting of: SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ
ID NO:34, SEQ laN0:35, SEQ NO:36, SEQ ID NO:37, SE() ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID
NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:4g, SEQ ID NO:49, SEQ 'ID NO:50, and SEQ ID NO:51, wherein the mature human leptin sequence has at least seven amino acid substitutions at positions where divergence is observed in corresponding positions in a leptin from another species.
102741 Embodiment 12. The chimeric polypeptide of any one of Embodiments 1-11, wherein a mature human leptin sequence comprises an amino acid sequence selected .from the group consisting of: SEQ 1D NO:12, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO2 1, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ
ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID
NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID
NO:45, SEQ ID NO:46, SEQ ID -NO:47,'SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:31, wherein the mature human leptin sequence has at least eight amino acid substitutions at positions where divergence is observed in corresponding positions in a leptin from another species.
[02751 Embodiment 13. The chimeric polypeptide of any one of Embodiments 1-12, wherein a mature human leptin sequence comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:12, SEQ ID NO:13, SEQ ID 'NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ NO:25, sw ID NO:26, SEQ ED NO:27, SEQ
ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID
NO:39, SEQ .11) NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ .10 NO:43, SEQ ID 140:44, SEQ
ID
NO:45, SEQ ID 140:46, SEQ ID .140:47, SEQ ID NO:48, SEQ ID 'NO:49, SEQ ID
NO:50, and =
SEQ 11) NO:51, wherein the mature human leptin sequence has at least nine amino acid substitutions at positions where divergence is observed in corresponding positions in a leptin from another species.
[02761 Embodiment 14. Thu chimeric polypeptide of any one of Embodiments 1-13, wherein a mature human leptin sequence comprises an amino acid sequence selected from the-Date Recue/Date Received 2021-11-12 =
group consisting of: SEQ ID NO: 2, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ED NO:27, SEQ
CD NO:34, SEQ ID NO:35, SEQ NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID
NO:45, SEQ ID NO:46, SEQ iD NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID .N0:50, and SEQ ID NO:51, wherein the mature human leptin sequence has at least. ten amino acid substitutions at positions where divergence is observed in eonTsponding positions in a leptin from another species.
[0277i Embodiment 15. The chimeric polypeptide of any one of Embodiments 1-14, wherein the mature human leptin sequence comprises the amino acid sequence of SEQ ID
NO:24.
[0278] Embodiment16. The chimeric polypeptide of any one of Embodiments I-
15, wherein the chimeric polypeptide comprises an amino acid sequence with at least 80% identity with an amino acid sequence selected from the group consisting of: SEQ ID
NO:29, SEQ ID
NO:30, SEQ ID .NO:32, SEQ ID NO:33, SEQ ID NO:52, SEQ ID .NO:53, SEQ ID NO:54, SEQ
ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID
NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID.
.NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ NO:71, SEQ
' ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID
NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, = SEQ ID NO:84, and SEQ ID NO:85.
102791 Embodiment 17. The chimcric polypeptide of any one of Embodiments 1-
16, wherein the chimeric polypeptide comprises an amino acid sequence with at least 80% identity with an amino acid sequence selected from the group consisting of: SEQ ID
NO:29, SEQ CD
NO:30, SEQ ID NO:32, SEQ ID NO:33.
102801 Embodiment IS. The chitneric polypeptide of any one of Embodiments 1-
17, wherein the chimeric polypeptide comprises an amino acid sequence with at least 80% identity = the amino acid sequence of SEQ ID .NO:33.

=
Date Recue/Date Received 2021-11-12 102811 Embodiment 19. The chimeric poly-peptide of any one of Embodiments 1-
18, wherein the chimeric polypeptide comprises an amino acid sequence with at least 90% identity the amino acid sequence of SEQ ID NO:33. .
[0282] Embodiment 20. . The chimeric polypeptide of any one of Embodiments 1-
19, wherein the chimeric polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ II) NO:29, SEQ ED NO:30, SEQ ID NO:32, SEQ H) NO:33, SEQ ID
NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO;57, SEQ ID
.NO:58, SEQ ID NO:59, SEQ ED NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ
ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID
NO:69, . SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID 'NO:73, SEQ ID NO:74, SEQ ED
NO:75, SEQ ID NO:76, SEQ ID NO:77, .SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, ED
SEQ ID NO:82, SEQ If) NO:83, SEQ ED NO:84, and SEQ ID NO:85.
[02831 Embodiment 21. The chimeric polypeptide of any one of Embodiments 1-
20, wherein the chimeric polypeptide comprises an amino acid sequence selected from the group .consisting of': SEQ ID NO:29, SEQ ED NO:30, SEQ ID NO:32, SEQ ID NO:33.
-102841 Embodiment 22, The chimeric polypeptide of any one of Embodiments 1-
21, wherein the chimeric polypeptide comprises the amino acid sequence of SEQ ID -N0:29.
= 102851 Embodiment 23. The chimeric polypeptide of any one of Embodiments 1-21, wherein the chimeric polypeptide comprises the amino acid sequence of SEQ ID
NO:30.
[0286] Embodiment 24. The chimeric polypeptide of any one of Embodiments 1-21, wherein the chimeric polypeptide comprises the amino acid sequence of SEQ ID
NO:32.
102871 Embodiment 25. The chimeric polypeptide of any one of Embodiments 1-21, wherein the chitneric polypeptide comprises the amino acid sequence of SEQ ID
NO:33.
102881 Embodiment 26. A method lin- treating a disease or disorder in a subject, comprising administering a chimeric polypeptidc of any one of Embodiments Ito 25 to a subject in need thereof in an amount effective to treat said disease or disorder.
102891 Embodiment 27. The method according to Embodiment 26, wherein the disease or disorder is. disease or disorder is selected from the group consisting of:
lipodystrophy, Date Recue/Date Received 2021-11-12 dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease, diabetes (including type 1 and type II), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAELD), metabolic syndrome X and Huntington's Disease.
102901 Embodiment 28. The method of Embodiment 26 or Embodiment 27, wherein the disease or disorder is lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease or diabetes.
102911 Embodiment 2.9. The, method of any one of Embodiments 26-28, wherein the disease or disorder is type I diabetes or type 11 diabetes.
[02921 Embodiment 30. The method of any one of Embodiments 26-23, wherein the disease or disorder is obesity.
=
102931 Embodiment 31. The method of any one of Embodiments 26-28, wherein the disease or disorder is lipodystrophy or leptin deficiency.
102941 Embodiment 32. A pharmaceutical composition comprising a chimeric polypeptide according to any one of Embodiments 1-25 and a pharmaceutically acceptable excipient.
IX. Informal Sequence Listing = 102951 An informal listing of sequences disclosed herein follows:
VPIQKVQDDTKILIKTIVTIIINDESHT-X aa-SVSSK.QM.VTGLDFIPGLEIPILTI.SKMDQTLA
VYQQILTSMPSRNVIQISNDLENLR.DLLHVLAFSKSCHLPQASGLETLESLGGVLEAS
GYSTEVVALSRLQGSLQDMLQQ1..DI.SPGC, wherein X.aa at position 28 is Q or absent (SEQ ID NO: I).
VPIQKVQDDTICILIKTIVTRINDISHTQSVSAKQRVTGLDFIPGLHPILSLSKNIDQTLAVY
QQVLTSLPSQNVLQIANDLENLILD111.1-111.11.AFSKSCSLNISGLOKPESLDGVI_EASLY
STEVVALSRLOGSLQDILQQ-LDVSPEC (SEQ ID NO:?).
VPIQKVQDDTKTLIKTIVTRINDISHTSVSAKQRVTGLDFIPGLHPILSLSKMDQTLAVYQ
QVIS.TS LPSQNVLQIANDLENLRIX. L.HL ESK SCS:L.PQTSCI.QK P ES ux; vLEAS1. S
TEVVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO:3).
Date Recue/Date Received 2021-11-12 MVPIQKVQDDIKTLIKTIVTRINDISFIT- Xaa-SVSSKQKVTG LDFIPOLIIPILTLSK MDQTL
A.VYQQILTS MPS RNVIQISNDLENIADLLHVLAFSKSCIELPQASGLE.TL ESLGG VLEA
SGYSTEVVALSRLQGSLQDMLQQLDI.SPGC, wherein Xaa at position 29 is Q or absent (SEQ ID NO:4).
MVPIQKVQ DDT KTLEKTIVTRI NDISHTQSVSAKQR.VIGLDFIPGLITPILSLSKMDQTLAV
YQQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDGVL EASL
YSTEVVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO:5).
MVPIQKVQDDTKTLIKTIVTRINDISHTSVSAKQRVTGLDFIPGIIIPILSLSKMDQTLAVY
QQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDGVLEASL Y
STEVVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO:6).
VPIWRVQDDTKILIKTIVTRISDISHMQSVSSKQRVTGLDFIPGLI-IPVLSLSKMDQTLAI Y
QQILTSLPSRNVIQISNDLENLRDLLYILLASSKSCPLPQARALETLESLGGVLEASLY S
TEVVALSRLQGALQDMLRQLDLSPGC (SEQ ID NO:7)..
= M VPIWRVQDDTKTLTKTIVTRISDISHMQSVSSKQRVTGLDFIPGLH PVLSLSK IvIDQTLAI, YQQILTSLPSRNVIQISNDLENLRDILHLLASSKSCPLPQARALETLESLGGVLEASLY
STEVVALSRLQGALQDMLRQLDLSPGC (SEQ ID NO:8).
,=
VPICKVQDDTKILIKTIVTRINDISFIT-Xaa-SVSSKQRVTG LDFIPGLIIPLLSLSKM DQTLA
= YQQILTS LPSRNVVQ1SNDLENLRDLLHLLAASKSCPLPQVRALESLESLGVVLEASL
YSTEVVALSRLQGSLQDNILRQLDLSPCiC, wherein :Xaa at position 28 is Q or absent (SEQ ID NO:9).
MVPICKVQDDTKTLIKTIVTRLNDISHT-Xaa-SVSSKQRVTGLDFIPGLHPLLSLSKMDQTL
Al YQQILTSLPSRNVVQ ISND LENLRDL L HLLAASKSCPL PQ VRA LESLESLGV V LEAS
LYSTEVVALSRLQGSLQDMLRQLDLSPGC, wherein Xaa at position 29 is Q or absent (SEQ ID NO:10).
MHWGTLCGIFLWLWPYLEYVOAVPIQKVQDDTKTLIKTIVTRINDISITTQSVSSKQKVIlli LDFI PGLHP ILTLSKM DQTLA V YQQ ILTSM PS RNVIQ ISNDLENLRD L LH V LA FSKSCH
LPWASGLEILDSLGGVLEASGY STEVVALSRLQGSLQDMLWQLDLSPGC (SEQ ID
NO: I I) 96 =
Date Recue/Date Received 2021-11-12 VPIQKVQDDIKTLIKTIVTRINDISH-Xaa-Xaa-SVSSKQKVTGLDFIPOLHPILILSKMDQT
LAVYQQILISMPSRNVIQISNDLENERDLLIIVLAFSKSCHI.PWA.SGLETLDSIGGVI.E
ASGYSTEVVALSRLQCSIQDMLWQEDI.SPGC, wherein: .Xaa at position 27 is T or A;
and Xaa at position 28 is Q or absent (SEQ ID NO:12).
MVPIQKVQDDTKTLIKTIVTRINDISH-Xaa-Xaa-SVSSKQKVTGI,DFIPGI.HPILTLSKMDQ
TLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETEDSLGGVI, EA.SCIYSTEVVALSRLQGSLQDMI.WQLDLSPOC, wherein: Xaa at position 28 is T or A;
and Xaa at position 29 is Q or absent (SEQ ID NO:13).
VPIQKVQSDTKTLIKTIVTRNDISHTQSVSSKQRVTOLDFIPGLHPVLTLSQMDQTLAIYQ
QIIIN.L.PSRNVIQISNDLENE,RDLIALL.A.FSKSCHL.PLASOLETLESI..GDVI,EASLYSTE
= WALSRLQGSLQDNILWQLDLSPCIC. (SEQ ID NO:14).
IviVPIQKVQSDTICTLIKTIVTRINDISHTQSVSSKQRVTGLDFIPOLHPVLTLSQMDQTLAI
YQQILJNLPSRNVIQISNDLENLRDLE,HLLAFSKSCfII,PI,ASGLETLESLGDVLEASLY -STEVVALSRLQGSLQDMLWQLDLSPGC (SEQ. ID NO:15).
VPDIKVQDDTKILIKTIVTRIIN-DISHTQSVSARQRVTGLDFIPGLHPILSESKMDQTLAVY
QQ-11,TS1,-PSQNVI.Q1AHDLENLRDLIIIIIAFSKSCSI.PQTRGLQK.PESI.DGVLEASLY
STEVVALSRLQGSLQD1LQQLDESPEC (SEQ. ID NO:16). =
MVPIHKVQDDTKTLIKTIVIRINDISHTQSVSARQRVTGLDFIPGLHPILSESKMDQTLAV
YQQILTSLPSQNVLQIAH:DLENLRDLLHLLAFSKSCSLPQTRGLQKPESLDGVLEASL
YSTEVVALSRLQGSLQDILQQLDESPEC (SEQ ID NO:17).
ISIEKIQADTKTETKIIITRIIQLSTQNGVSTDQRVSOLDFIPGNQQFQNLADMDQTLAVYQ
QILSSLPMPDRTQISNDLENERSLEALLAILKNOPETRSDGLDTMEIWGGIVEESLYST
EVVTLDRERKSLKNIEKQLDHIQG (SEQ ID NO:18).
MRCILLYGELCVWQFILYYSIIPISIEKIQA DTKTI_TKTUTRI IQ ESTQNCIVST DQ R VSOLDE
IPGNQQFQNLADMDQTLAVYQQ1LSSLPMPDRTQISNDLENLRSLFALL,ATLKNCPFT
RSDOLDTME1WGG1.VEESLYSTEVVTLDRERKSLKNIEKQLDHIQG (SEQ ID NO:19).
VPIQKVQDDTKT.I.,IKTEVTRIND1SIITQSVSSKQKVIGI.DFIPG1:1-1PIETESKMDQTLAVY

STEVVALSRLQGSLQDMLWQLDLSPGC (SEQ ID NO:20).

Date Recue/Date Received 2021-11-12 VPIQKVQDDTKTLIKTEVTRINDISHAQSVSSKQKVTGLDFIPOLHP I LTLS KMDQTLAVY
QQ1LTSMPSRNVIQISNDLENI,RDLIIIVLAFSKSCHLPWASGLETI,DSLGGVLEASG
YSTEVVALSRLQGSLQDMLWQLDLSPGC (SEQ ED NO:2I ).
VINKVQDDTKTLIKTIVTRINDISHTSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQ
QILTSMPSRNVIQISNDLENLRDLLI-IVLAFSKSCHLPWASGLETI,DSLGGVI,EASGYS
TEVVALSRLQGSLQDWILWQLDLSPGC (SEQ ID NO:22).
VPIQKVQDDTKTLIKTIVTRINDISHASVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQ
QII.TSMPSRNVIQISNIKENLIIDLLEIVI,AFSKSCIII,PWASGLETLDSI,GGVLEASGYS
TEVVALSRLQGSLQDNILWQLDLSPGC (SEQ ID NO:23).
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAV
YQQILTSMPSRNVIQISNDLENLRDI,L.HV LAFSKSCRI,PWASCiLEILDSLGGVLEASG
.YSTEVV.ALSRLQGSLQDMLWQLDLSPGC. (SEQ ID NO:24).
IvIVI'IQKVQDDTICTLIKTIVTRINDISHAQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAV
=
YQQILTSNIPSRNVIQISNDLENLRDLLFIVLAFSKSCHLPWASCILEILDSLGGVLEASC;
YSTENNALSRIQGSLQDMLWQLDLSPGC. (SEQ ID NO:25).
MVP IQKVQDDIKTL IK TI VTRINDISHTS VSSKQKVTGLDFI PGLH P ILTLS K.14.4 DQTLA V Y

STEVVA LSRLQGSLQDMLWQLDLSPOC (SEQ ID NO:26).
MVPIQKVQDDTKILIKTEVTRINDISHASVSSKQKVICILDFIPOIIIPILTLSK.MDQTLAVY
QQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGY
STEVV.kLSRLQGSLQDMLWQLDLSPGC (SEQ ID NO:27.).
PIQR.VQDDTKTL.IKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQ
ILTSLQSRSVVQEANDLANLRALLRLLASAKSCPVPRARGSDTIKGLGNVLRASVHST
= EVVALSRLKAALQDMLR.Q1DRNPO(; (SEQ ID NO:28), PIQRVQDDTKTLEKTIITRINDISPPOGVCSRPRVAGL.DFIPRVQSVRILSGIvIDQ11,ATYQQ
ILTSLQSFLINTIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKOLGNVLIZASVHSTE
VVALSRLKAALQDNILR.QI.DRNPGC (SEQ ID NO:29).

Date Recue/Date Received 2021-11-12 PIQRVQDDIKTLIKT.IITRINDISPPQOVSSRPRVAGLDFIPRVQSVRTLSGIVIDQTLATYQQ1 LTSLQSRNVIQISNDLENLIZDLLUIVLAFSKSCPVPRAR.GSDTIKOLGNVLRA.SVFISTE
VVALSELLKAALQDWILRQLDRNPOC (SEQ ID .NO:30).
MPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQ
QILTSLQSRSVVQ1ANDLANI.RALLRLLASAKSCPVPIZARGSDIIK.GLONVLRASVHS
= TEVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:31).
MPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQ -QII.TS[,QSRNVIQISNDLENI.,RDI.1_I-IVLA.FSKSCPVPRAROSDTIKGI..GNVL;RASVI-IST
EVVALSRLKAALQDMIAQI.ORNPGC (SEQ ID NO:32). .
. MPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMD.Q1LATYQ
QIUSLQSRNVIQISNDLENLIWILHVLAFSKSCI'VPRAROSDTIKOLONVLRASVHST
EVVALSRIKAALQDMLRQEDRNPGC (SEQ ID NO:33).
NIDKTHTCPPCPAPELLGGPSVELFPPKPKDTLMISRTPEVTC'VVNIDVSHEDPEVIUNWY
'MOVEN/1E1NA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALI}A
= KTISKAICGQPREPQVYTLPPSRDELTICNIQVSLTONKGFYPSDIAVEWESNGQPENN
YKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMFIEALIINHYTQKSLSII.SPG
KVPIQKVQ DDTKTLIKTTVIRINDISHTQSVSSKQKVTGLDFIPOL WILMS PiEDQTL
. _ A.VYQQILTSNIPSRNVIQISNDLENIADLI...HVLAFSKSCI-ILPWASG LETLDSLGOVLEA
SGYSTEVVALSRLQGSLQDIVfLAVQ.LDLSPGC (SEQ ID NO:34) MVPIQKVQDDTKTLIKTIVTRINDISHIQSVSSKQKVTGLDFIPOLHPILTLSKAIDQT LA V
VOO11.:TSMPSRNVIQI.SNDLEN.L.RDI,LIIVI_AFSKSCHLPQASOLETIDSLOGNILEASCI
YSTEVVALSRLQGSLQDMLQQLDLSPOC (SEQ ID NO:35).
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGI_HPILTLSKMDQTLAV
YQQILTSMPSRNVIOICNDLENIADLLHVLAFSKSCHIPWASGLETLDSLOGVLEASO
YSTEVVALSRLQGSLQDMLWQLDLSAGC (SEQ ID NO: 36).
NIVPIQKVQDDIKTLIKTIVTRINDISHIQSVSSKQKVTGLEEIPGLHPILTLSKAIDQTLAV
= YQQ I LTS MP SRNV IQISND LE NLIt DL LH V LA FSK SC H 1_11QA SOL ET LES
LGG V LE ASG
YSTEVVALSRLQGSLQDMLQQLDLSAGC (SEQ ID NO:37).

Date Recue/Date Received 2021-11-12 =
MVPIQKVQDDTICTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKNIDQTLAV =
= YQQILTSNIPS.RNVIQISNDLENEADLLHVLAFSKSCSLPQASGLETLESLGEVLEASGY
STEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 38).
NIVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTISKAIDQTLAV
YQQILTSMPSRNVIQISNDLENLRDLLHVIAFSKSCSLPQASGLETLDSLGGVLEASG
YSTEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 39).
NIVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAV
YQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLDSLGF.VLEASG
YSTEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 40).
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAV
'YQQILTSMPSRNVIQIZSNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGEVLEASG
YSTEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 41).
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLEIPILTLSKMDQTLAV
YQQIILTSNIPSRN S NDLENLRDLLHVLAFSKSCS PQASGLETL DSLGG L EASG
YSTEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 42).
MVPIQKVQDDTKTI. IKTIVTRINDISHTQSVSSKQKVTGLDFI PGLH P.I LUSK M DQTLA V
YQQICISMPSRNVIQISNDLENLRDLLIIVLAFSKSCHLPQASGLETLDSLGEVLEASG
YSTEVVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO: 43).
NIVPIQKVQDDTKTLIKTIVTRINDISIITQSVSSKQKVTGLDFIPGLIIPILTLSKMDQTLAV =
YQQILTSMPSRNVIQISNDLENLR.DLLHVLAFSKSCSLPQTSGLETLDSLOGVLE,ASG
YSTEWALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 44).
MVPIQI.(VQDDTKTLIK TIVTRINDIS IFIQSVSSKQICVTGLDFLPGLI-IPI LTLS.KMDQTLA
YQQILTSMPSRNVIQISNDLENLRDLLIIVLAFSKSCSLPQASGLETLESLGEVLEASGY
STEVVALSRLQGS LQDMLWQLDLSPEC (SEQ ID NO:.45).

YQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASOLETLDSLGGVLEASG
YSTEVVALSRLQGSLQDILQQLDLSPEC (SEQ IDNO: 46).

=
Date Recue/Date Received 2021-11-12 . .
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAV
YQQIILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGOVLE.ASG
YSTE VVALSRLQGSLQDILQQLDVSP EC (SEQ ID NO: 47).
NIVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKIvIDQTLAV

YSTEVVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO: 48).
=
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLIIPILTLSKIVIDQTLAV
YQQILTSMPSRNVIQISNDLENLRDITHVLAFSKSCSLPQTSGLETLDSLGEVLEASGY
STEVVALSRLQGSLQDMLWQLDLSPEC (SEQ ID NO: 49).
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLA V
.YQQ1LISMPSRNVIQISNDLENLRDLLIIVLAFSKSCHLPQASGLE:TLDSLGEVLEASG
YSTEVVALSRLQGSLQDMLQQLDLSPGC (SEQ ID NO: 50).
MVPIQKVQDDIKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPOLHPILTLSKMDQTLAV
YQQILTSMPSRNVIQISNDLENLRDLLHVLAPSKSCHLPQASGLETLDSLGE VLEASG
YSTEVVALSRLQGSLQDMLQQLDLSPEC (SEQ ID NO: 51).
PIQKVQDDTKTLIKTIVTRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQ1LATYQ .
QILTSLQSRSVVQIANDLANLRALLRLLASAKSCPVPRA RGSDTIKGLGNVLRASVHS =
TEVVALSRLKA.ALQDMLRQLDR.NPGC (SEQ ID NO:52).
MP IQKVQDDTKTLIKTIVTRINT)ISPPQGVCSRPRVAGLDFIPR.VQSVRTLSGMDQILATY
QQILTSLQSRSVVQIANDLANLRALLRLLASAKSCPVPRARGSDTIKGLGNVLMSV
HSTEVVALSRLKAALQDIALRQLDRNPGC (SEQ ID NO:53).
PIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPIRVQSVRTLSK.MDQTLAVYQ

TEVVALS:RLKA.ALQDMLRQLDR.NPGC (SEQ ID NO:54).
MPIQRVQDDTKTLIKTIITRINDISPPQ(3VCSRP.R.VA.GLDFIPRVQSVRTLSKMDQTLAVY
QQILISLQSRSVVQIANDLANLRALLRLLASAKSCPVPRARGSDTIKGLGNVLRASV
.1-1STEVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:55).

=
Date Recue/Date Received 2021-11-12 PIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQ
ILTSLQSRSVVQIANDLANLRALLRLLASAKSCPVPRARGSDTIK.GLGNVLRASVHST
EVVALSRLQGSLQDMI.WQ1-DLNPGC (SEQ ID NO:56).
MP IQRVQDDTKTLIKTETRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILAT YQ

TEVVALSRLQGSLQDMLWQLDLNEGC (SEQ ID NO:57).
PIQRVQDDTKTLFICITITRNDISHTQSVSSKQKVTGLDFIPGLHPILTLSGMDQILATYQQI
LTSLQSRSVVQ1ANDLANI.RALLRLLASAKSCPVPRARGSDTIKGLGNVI_RASVII ST
EVVALSRLKAALQDMLRQLDRNPGC (SEQ ID 'NO:581.

QIL'F'SLQSRSVVQlANDLANLRALLRLLASAKSCPVPRARGSDTIKGLGNVLRASVHS
TEVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:59).
PIQRVQDDTKTLIKTIITRNDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQ
ILTSLQSRSVVQIANDLANLRALLRLLASAKSCHLRWASGLEILDSLGGVLEASGYST
EVVALSRLICAALQDMLRQLDRNPGC (SEQ ID NO:60).
M.P1 QRVQDDTKTLIKTIITR INDISPPQGVCSRPR VAG LDFIPR VQSVRTLSGMDQI LATYQ

STEVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:61).
PIQKVQDDTKTLIKTIVTRINDISPPQGVCSRPRVAGEDFIPRVQSVRTLSG M DQII-ATYQ
QILTSLQSRNITIQISNDLENLRDELHVIAFSKSCPVPRARGSDTIKGLGNVLRASVHST
EVVALSRLKAALQDMLRQLDR-NPGC (SEQ ID NO:62) MPIQK.VQDDTKILIKTIVTRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATY

STEVVALSRLKAALQDMIAQLDRNPGC (SEQ ED NO:63) PIQRVQDDIKTLIKTIITRINDISIITQSVSSK.QKVTGLDFIPGI,HPILTLSGMDQILATYQQ1 LTSLQSRNVIQISNDLENLRD. LIAVLAFSKSCPVPRARGSDTIKOLGNVIRASVHS __ FE
VVALSRLKAALQDMILRQLDRNPGC (SEQ ED NO:64) Date Recue/Date Received 2021-11-12 QILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVH.ST
EVVALSRLKAALQDMLRQLDRNPGC (SEQ :ID NO:65) PIQRVQDDTKTLIKTLITRDIDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQ
ILTSLQSRNVIQISNDLENLRDLUIVLAFSKSCHLPWASGLETLDSLGGVLEA.SGYST
. EVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:66) _ = MPIQRVQDDTKTLIKTITTRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQ
QILTS.I.QSRNVIQISNDLENLRDLLINLAFSKSCHLPWASGLETLDSLGGVLEA.SOYS
TEVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:67) = P1QRVQDDTKTLIKTIITRINDISHTQSVSSKQKVTGLDFLPGLHPILTLSGMDQILATYQQ1 LTSLQSRSV VQEANDLANLRALLRLLASAKSCPV PRARGSDTIKOLGN VLRAS 1-1S.1' EVVALSRLQGSLQDMLWQLDLNPGC (SEQ ID NO:68) MPIQRVQDDTKTLIKTIITRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSGMDQELATYQ
QILTSLQSRSVVQIANDLANLRALLRLLASAKSCPVPRARGSDTIIKGLGNVLRASVHS
TEVVALSRLQGSLQDMLWQLDLNPGC (SEQ ID NO:69) =

EVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:70) MPIQR.VQDDTKTLIKTIITRINDISIITQSVSSKQ.KVTGLDFIPGLIIPILTI.SGNIDQILATYQ
QILTSLQSRSVVQIANDLANLRALLRLLASAKSCHLPWASGLEILDSLGGVLEASGY.
STEVVALSRLKAALQDIALRQLDRNPGC (SEQ ID NO:71) =
PIQR.VQDDTKTLIKTIITRINDISHTQSVSSKQKVTGLDFIPGLIIPILTLSGMDQII,ATYQQ-1 .LTSLQSRNVEQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTE , VVAE,SRLK.AALQDMI.ROLDRN.PCIC (SEQ ID NO:72) MP EQR.VQ DDTKTLIKTIITRINDISIITQSVSSKQKVTGLDFIPGLHPILTLSGMDQILATYQ
QILTSLQPNVIQISNDLENLRDLLI-IVLAFSKSCHLPWASGLETLDSLGGVLEASGYS
TEVVALSRLKAALQDMI:RQLDRNPGC (SEQ ID NO:73).

' =
Date Recue/Date Received 2021-11-12 PIQKVQDDTKTLIKTIVTRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQ
QILTSLQSRSVVQIANDLANLRALLRLLA.SAKSCPVPRARGSDTIKGLGNVLRASVHS
TEVVALSRLKAALQDMLIZQLDRNPGC (SEQ NO:74).
MPIQKVQDDTKTLIKTIVT RINDI SPPQGVSS RPRV A GLD FI PR V QSV LSG M DQI LA T Y
QQILTSLQSRsVVQEANDLA:NLRAI..LRLLASAXSCPVPRARGSDTIKGLGNVLRASV
HSTEVVALSRLKAALQDMLRQLDRNPGC (SEQ [D NO:75).
PIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSKMDQTLAVYQQ
ILTSLQSRSVVQIANDLAN'LRALLRLLASAKSCPVPRARGSDTFKGLGNVLRASVHST
EVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:76). =
MPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSKMDQTLAVY
QQILTSLQSRSVVQ[ANDLANLRALLRLLASAKSCPVPRARGSDTIKGLGNVLRASV
HSTEVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:77).
= PIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQI
LTSLQSRSVVQIANDLANLRALLRELASAKSCPVPRARGSDTIKGLGNVLRASVIIST
EVVALSRLQGSLQDMLWQLDLNPGC (SEQ ID NO:78), MPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVACILDFIPRVQSVRTLSGMDQILATYQ
QILTSLQSRS VVQIAND LAN LIZ ALLRLLAS AKSCP V MAROS DIRG G N VLIt AS V I1S
TEVVALSRLQGSLQDMLWQLDLNPGC (SEQ ID NO:79).
PIQRVQDDTKILEKTIITRINDISPPQGVSSRPRVAGLDEEPRVQSVR.TLSGMDQILATYQQ1 LTSLQSRSVVQ1A,NDLANLRALLRLLASAKSCHLPWASGLETLDSLOGVLEASGYST
EVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:80).
MPIQRVQDDTKTUKTIFIR.INDISPPQGVSSRPR.VAGLDF[PRVQSVRTII.SGMDQILATYQ
QILTSLQSRSVVQ1ANDLANLRALLRLLASAKSCHLPWASGLEILDSLCiGVLEASGY
STEVVALSRLK.AALQDMLRQII.DRNPGC (SEQ ID 'NO:81).
PIQKVQDDTKTLIKTIVTRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGIvIDQ[LATYQ
QILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHST
EVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO:82) Date Recue/Date Received 2021-11-12 MPIQKVQDDTKTLIKTEVTRTNDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGIVIDQILATY
QQILISLQSRNVIQISNDLENI.RDLLEIVLAFSKSCPVPRARGSDTIKGLONVLRASVI-1 STEVVALSRLKAALQDMIRQI..DRNI'Cie (SEQ NO:83) =
. PIQRVQDDTKTLIKTIITRENDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQ1 :LTSLQSRNVIQISNDLENIADLLEIVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTE
WALSRLICAALQDMIRQLDRNTGC (SEQ ID NO:84) IAPIQRVQDDTKTLIKTUTRINDISPPQOVSSRPRVAGLDFIPRVQSAIRILSOMDQILATYQ
QII.TSLQSRNVIQISNDLENERDI.1,1-EVIAFSKSCHLPWASGIETI,DSI.G0VII.EASGYS
TEVVALSRLKAALQD/vILRQLDIINPGC (SEQ ID NO:85) KCNTATCATQRLANFINRSSNNLGPVLPFINVGSNTY (SEQ ID NO:86);
KCNTATCATQUANFLVHSSNNEGAILSS'INVGSNTY (SEQ ID N.0:87);
KCNTATCATQRLANFLVHSSNNEGPILPPINVGSNTY (SEQ ID NO:88).
CGNESTCIALGTYTQDFNKFHTFPQTAIGVGAP (SEQ ID NO:89);
CSNLSICVLGKLSQELKKLQTYPRTNTGSGIP (SEC) ID NO:90);
KCNTATCVLGRLSQELHRLQTYPRTNIGSNTY (SEQ ID NO:91). , X'-Xaal-Cys2-Asn3-Thr4-Ala5-Thr6-Cys7-Alat-Thr9-Glej-Argli-Leu12-Alail-Asn14-Phel5-Letti6-Val17-11is"-Seri9-Ser20- Xaa21-Asn"-Phe23- Xaa"- Xaa"- Xaa1.6- Xaa27- Xaa23 "-Thr3"-Xaa31-Va132-Gly33-Ser"-Asn35-Thr36-Ty137-X (SEQ ID NO:92) CNTATCATQRLANELVRSSNNLGPVLPFINVGSNIY-NI12 (SEQ ID NO:93) KCNTATCATQRLANFLVRSSKNLGPVLPPTNVGSNTY-NH2 (SEQ ID NO:94) CNTATCATQRLANFLNRSSKNLGPVLPPTNTVGSNTY-NH2 (SEQ ID NO:95) KCNTATCATQRLANFLVRSSNNLGPKLPPTNVGSNTY-NH2 (SEQ ID NO:96) CNTATCATQRLANFINRSSNNI.GPKLP.PINVGSNIY-N112 (SEQ ID NO:97) KCNTATCATQRLANFINRSSNNI.GPVE,PPTKVGSNTY-N1-12 (SEQ ID NO:98) CNTATCATQRLANFLVRSSNNLGPVLITIKVGSNIY.-NI-i2 (SEC) ID NO:99) KCNTATCATQRLANFLVEISSNNEGPILPPINVGSNTY-N112(SEQ ID .N 0: I 00) =
CNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY-NH2 (SEQ ID NO:10 I) Date Recue/Date Received 2021-11-12 CNTATCATQRLANFINHSSKNEGPELPPINVGSNTY-NH, (SEQ ID NO:102) CNTATCATQRLANFLVHSSNNFGPKLPPTNVGSNTY-NH2 (SEQ ID NO:103) CNTATCATQRLANFLVHSSN'NFGPLLPPTKVGSNTY-NH2(SEQ ID NO:1 04) CNTATCATQR.LANFINIISSNNEKPILPPINVGSNTY. -NI-12 (S EQ ED NO:105) .
CNTATCATQRLANFLVHSSNNEGKILPPINVOSISITY-NH2(SEQ ID NO:106) CNTATCATQRLANFLVI4SSNNEGPIKPIYINVG5NTY-N112(SEQ ID NO: I07) CNTATCATQRLANFLYFISSNNEGPILKRINVGSNIY-NH2(SEQ ID NO:108) -CNTATCATQRLANFLVHSSNNTGPILPKINVGSNTY-N112(SEQ ID NO:109) =;-Date Recue/Date Received 2021-11-12

Claims (10)

Claims:
1. A chimeric polypeptide comprising a wild type seal leptin polypeptide wherein at least one contiguous region of 1-30 amino acids of a wild type seal leptin sequence has been replaced with a contiguous region of 1-30 amino acids of a mature human leptin sequence, and wherein the chimeric polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 70.
2. The chimeric polypeptide of claim 1, wherein the chimeric polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID
NO: 70.
3. The chimeric polypeptide of claim 1, wherein the chimeric polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID
NO: 70.
4. The chimeric polypeptide of claim 1, wherein the chimeric polypeptide comprises the amino acid sequence of SEQ ID NO: 70 or 71.
5. Use of a chimeric polypeptide of any one of claims 1 to 4 for treating a disease or disorder in a subject, wherein the disease or disorder is lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease, type I diabetes, type II diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X or Huntington's Disease.
6. Use of a chimeric polypeptide of any one of claims 1 to 4 in the manufacture of a medicament for treating a disease or disorder in a subject, wherein the disease or disorder is lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease, type I diabetes, type II diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X or Huntington's Disease.
7. The use of claim 5 or 6, wherein the disease or disorder is lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease, type I diabetes or type II
diabetes.

Date Recue/Date Received 2021-11-12
8. The use of any one of claims 5-7, wherein the disease or disorder is obesity.
9. The use of any one of claims 5-7, wherein the disease or disorder is lipodystrophy or leptin deficiency.
10. A pharmaceutical composition comprising a chimeric polypeptide according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient.

Date Recue/Date Received 2021-11-12
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